Compositions and methods for organ specific delivery of nucleic acids

ABSTRACT

The present disclosure provides compositions which shown preferential targeting or delivery of a nucleic acid composition to a particular organ. In some embodiments, the composition comprises a steroid or sterol, an ionizable cationic lipid, a phospholipid, a PEG lipid, and a permanently cationic lipid which may be used to deliver a nucleic acid.

This application is a continuation of U.S. application Ser. No.17/191,895, filed Mar. 4, 2021, which is a continuation of InternationalApplication No. PCT/US2019/049565, filed Sep. 4, 2019, which claims thebenefit of priority to U.S. Provisional Application Ser. No. 62/726,741,filed on Sep. 4, 2018, the entire contents of each of which are herebyincorporated by reference.

This invention was made with government support under grant numberCA150245 and CA190525 awarded by The National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND 1. Field

The present disclosure relates generally to the field of molecularbiology. More particularly, it concerns tissue specific delivery of atherapeutic agent such as a nucleic acid, a protein, or a small moleculetherapeutic agent in lipid nanoparticles.

2. Description of Related Art

The CRISPR/Cas (clustered regularly interspaced short palindromicrepeat/CRISPR-associated protein (Cas)) technology can edit the genomein a precise, sequence dependent manner, resulting in a permanentchange. Because of the ability to target disease causing mutations, itholds incredible promise for one-time cures of genetic diseases. Todate, successful editing has been mediated mainly by viral vectors,which require laborious customization for every target and presentchallenges for clinical translation due to immunogenicity, generation ofantibodies that prevent repeat administration, and concerns about rarebut dangerous integration events. There remains a clear need toaccomplish CRISPR/Cas editing via synthetic nanoparticles (NPs) toexpand the safe and effective applications of gene editing.

CRISPR/Cas enables sequence-specific DNA editing by the RNA-guidedCRISPR-associated protein 9 (Cas9) nuclease, or its homologs, that formsdouble-strand breaks (DSBs) in genomic DNA. Cas9 is guided byprogrammable RNA called single guide RNA (sgRNA). The Cas9/sgRNA complexrecognizes the complementary genomic sequence with a 3′ protospaceradjacent motif (PAM) sequence. Following DNA cleavage, DSB repairpathways enable directed mutagenesis, or insertions/deletions (indels)that delete the targeted gene. For therapeutic utility, transient Cas9expression is preferred to limit off-target genomic alteration. Becauseboth Cas9 protein and sgRNAs must be present in the same cells,co-delivery of Cas9 mRNA and sequence targeted sgRNA in one NP is anattractive method, particularly for in vivo use where tissue penetrationand cellular uptake is more challenging. CRISPR/Cas editing usingviruses, membrane deformation, ribonucleoprotein complex delivery, andhydrodynamic injection are functional, but have limitations that couldhinder in vivo therapeutic use in the clinic, including persistentexpression of Cas9 and off target editing. Furthermore, these deliverysystem generally are not selective for the specific organs in whichediting is needed. For example, most lipid nanoparticles accumulatethrough the biological processes in the liver thus reducing the efficacyof the composition on delivery into the target organ.

Similarly, other therapeutic agents such as proteins and small moleculetherapeutic agents could benefit from organ specific delivery. Manydifferent types of compounds such as chemotherapeutic agents exhibitsignificant cytotoxicity. If these compounds could be better directedtowards delivery to the desired organs, then less off target effectswill be seen.

Therefore, there remains a need to develop new lipid nanoparticles whichshow preferential delivery to specific organs.

SUMMARY

In some aspects, the present disclosure provides lipid compositionswhich show organ specific delivery of the lipid composition. Thesecompositions may be used to deliver a nucleic acid component to aspecific organ.

In some aspects, the present disclosure provides compositionscomprising:

(A) a therapeutic agent; and

(B) a lipid nanoparticle composition comprising:

(1) a selective organ targeting compound;

(2) an ionizable cationic lipid; and

(3) a phospholipid;

wherein the composition preferentially delivers the nucleic acid to atarget organ selected from the lungs, the heart, the brain, the spleen,the lymph nodes, the bones, the bone marrow, the skeletal muscles, thestomach, the small intestine, the large intestine, the kidneys, thebladder, the breast, the liver, the testes, the ovaries, the uterus, thespleen, the thymus, the brainstem, the cerebellum, the spinal cord, theeye, the ear, the tongue, or the skin. In some embodiments, the targetorgan is selected from the lungs, the heart, the brain, the spleen, thelymph nodes, the bones, the bone marrow, the skeletal muscles, thestomach, the small intestine, the large intestine, the kidneys, thebladder, the breast, the testes, the ovaries, the uterus, the spleen,the thymus, the brainstem, the cerebellum, the spinal cord, the eye, theear, the tongue, or the skin.

In some embodiments, the target organ is the lungs, the lymph nodes, orthe spleen. In some embodiments, the target organ is the lungs. In otherembodiments, the target organ is the spleen. In other embodiments, thetarget organ is the liver. In other embodiments, the target organ is thelymph nodes.

In some embodiments, the selective organ targeting compound is apermanently cationic lipid. In some embodiments, the permanentlycationic lipid is present in a molar percentage of the lipidnanoparticle composition from about 5% to about 20%. In someembodiments, the molar percentage of the permanently cationic lipid ispresent from about 12% to about 18%. In some embodiments, the molarpercentage of the permanently cationic lipid is about 15%. In someembodiments, the permanently cationic lipid is present in a molarpercentage of the lipid nanoparticle composition from about 20% to about65%. In some embodiments, the molar percentage of the permanentlycationic lipid is present from about 40% to about 61%. In someembodiments, the molar percentage of the permanently cationic lipid isabout 50%.

In some embodiments, the permanently cationic lipid comprises aquaternary ammonium ion. In some embodiments, the permanently cationiclipid is further defined as:

wherein:

-   -   R₁ and R₂ are each independently alkyl_((C8-C24)),        alkenyl_((C8-C24)), or a substituted version of either group;    -   R₃, R₃′, and R₃″ are each independently alkyl_((C≤6)) or        substituted alkyl_((C≤6));    -   X⁻ is a monovalent anion.

In some embodiments, R₁ is an alkenyl_((C8-C24)) or substitutedalkenyl_((C8-C24)). In some embodiments, R₂ is an alkenyl_((C8-C24)) orsubstituted alkenyl_((C8-C24)). In other embodiments, R₁ is analkyl_((C8-C24)) or substituted alkyl_((C8-C24)). In other embodiments,R₂ is an alkyl_((C8-C24)) or substituted alkyl_((C8-C24)). In someembodiments, R₁ and R₂ are both the same. In some embodiments, R₃, R₃′,and R₃″ are each identical. In some embodiments, R₃, R₃′, and R₃″ areeach methyl. In some embodiments, X⁻ is halide anion such as bromide orchloride. In some embodiments, the permanently cationic lipid is furtherdefined as:

In other embodiments, the permanently cationic lipid is further definedas:

wherein:

-   -   R₄ and R₄′ are each independently alkyl_((C6-C24)),        alkenyl_((C6-C24)), or a substituted version of either group;    -   R₄″ is alkyl_((C≤24)), alkenyl_((C≤24)), or a substituted        version of either group;    -   R₄′″ is alkyl_((C1-C8)), alkenyl_((C2-C8)), or a substituted        version of either group; and    -   X₂ is a monovalent anion.

In some embodiments, R₄ is alkyl_((C6-C24)) or substitutedalkyl_((C6-C24)) such as octadecyl. In some embodiments, R₄′ isalkyl_((C6-C24)) or substituted alkyl_((C6-C24)) such as octadecyl. Insome embodiments, R₄″ is alkyl_((C≤24)) or substituted alkyl_((C≤24)).In some embodiments, R₄″ is alkyl_((C≤8)) or substituted alkyl_((C≤8))such as methyl. In some embodiments, R₄′″ is alkyl_((C1-C8)) orsubstituted alkyl_((C1-C8)) such as methyl. In some embodiments, X₂ is ahalide such as chloride or bromide. In some embodiments, the permanentlycationic lipid is further defined as:

In some embodiments, the permanently cationic lipid is further definedas:

wherein:

-   -   R₁ and R₂ are each independently alkyl_((C8-C24)),        alkenyl_((C8-C24)), or a substituted version of either group;    -   R₃, R₃′, and R₃″ are each independently alkyl_((C≤6)) or        substituted alkyl_((C≤6));    -   R₄ is alkyl_((C≤6)) or substituted alkyl_((C≤6)); and    -   X⁻ is a monovalent anion.

In some embodiments, R₁ is an alkenyl_((C8-C24)) or substitutedalkenyl_((C8-C24)). In some embodiments, R₂ is an alkenyl_((C8-C24)) orsubstituted alkenyl_((C8-C24)). In other embodiments, R₁ is analkyl_((C8-C24)) or substituted alkyl_((C8-C24)). In other embodiments,R₂ is an alkyl_((C8-C24)) or substituted alkyl_((C8-C24)). In someembodiments, R₁ and R₂ are both the same.

In some embodiments, R₃, R₃′, and R₃″ are each identical such as R₃,R₃′, and R₃′ are each methyl. In some embodiments, R₄ is alkyl_((C≤6))such as ethyl. In some embodiments, X⁻ is halide anion such as bromideor chloride.

In some embodiments, the permanently cationic lipid is further definedas:

In other embodiments, the selective organ targeting compound is apermanently anionic lipid. In some embodiments, the permanently anioniclipid is present in a molar percentage of the lipid nanoparticlecomposition from about 5% to about 50%. In some embodiments, the molarpercentage of the permanently anionic lipid is present from about 10% toabout 45%. In some embodiments, the molar percentage of the permanentlyanionic lipid is about 30%. In some embodiments, the permanently anioniclipid comprises a phosphate group.

In some embodiments, the permanently anionic lipid is further definedas:

wherein:

-   -   R₁ and R₂ are each independently alkyl_((C8-C24)),        alkenyl_((C8-C24)), or a substituted version of either group;    -   R₃ is hydrogen, alkyl_((C≤6)), or substituted alkyl_((C≤6)), or        —Y₁—R₄, wherein:        -   Y₁ is alkanediyl_((C≤6)) or substituted alkanediyl_((C≤6));            and        -   R₄ is acyloxy_((C≤8-24)) or substituted acyloxy_((C≤8-24)).

In some embodiments, R₁ is an alkenyl_((C8-C24)) or substitutedalkenyl_((C8-C24)). In other embodiments, R₂ is an alkenyl_((C8-C24)) orsubstituted alkenyl_((C8-C24)). In other embodiments, R₁ is analkyl_((C8-C24)) or substituted alkyl_((C8-C24)). In other embodiments,R₂ is an alkyl_((C8-C24)) or substituted alkyl_((C8-C24)). In someembodiments, R₁ and R₂ are both the same.

In some embodiments, R₃ is hydrogen. In other embodiments, R₃ is —Y₁—R₄,wherein:

-   -   Y₁ is alkanediyl_((C≤6)) or substituted alkanediyl_((C≤6)); and    -   R₄ is acyloxy_((C≤8-24)) or substituted acyloxy_((C≤8-24)).

In some embodiments, Y₁ is substituted alkanediyl_((C≤6)) such as2-hydroxypropanediyl. In some embodiments, R₄ is acyloxy_((C≤8-24)) suchas octadecenoate. In some embodiments, the permanently anionic lipid isfurther defined as:

In other embodiments, the selective organ targeting compound is a C₆-C₂₄diacyl phosphotidylcholine. In some embodiments, the diacylphosphotidylcholine is present in a molar percentage of the lipidnanoparticle composition from about 5% to about 50%. In someembodiments, the molar percentage of the diacyl phosphotidylcholine ispresent from about 10% to about 45% such as about 30%.

In some embodiments, the selective organ targeting compound comprises atleast two fatty acid chains, a quaternary amine, and an anionicphosphate group. In some embodiments, the diacyl phosphotidylcholine isfurther defined as:

wherein:

-   -   R₁ and R₂ are each independently alkyl_((C8-C24)),        alkenyl_((C8-C24)), or a substituted version of either group;    -   R₃, R₃′, and R₃″ are each independently alkyl_((C≤6)) or        substituted alkyl_((C≤6)); and    -   X⁻ is a monovalent anion.

In some embodiments, R₁ is an alkenyl_((C8-C24)) or substitutedalkenyl_((C8-C24)). In some embodiments, R₂ is an alkenyl_((C8-C24)) orsubstituted alkenyl_((C8-C24)). In other embodiments, R₁ is analkyl_((C8-C24)) or substituted alkyl_((C8-C24)). In other embodiments,R₂ is an alkyl_((C8-C24)) or substituted alkyl_((C8-C24)). In someembodiments, R₁ and R₂ are both the same.

In some embodiments, R₃, R₃′, and R₃″ are each identical. In someembodiments, R₃, R₃′, and R₃″ are each methyl. In some embodiments, X⁻is halide anion such as bromide or chloride. In some embodiments, thediacyl phosphotidylcholine is further defined as:

In some embodiments, the ionizable cationic lipid is present in a molarpercentage of the lipid nanoparticle composition from about 5% to about30%. In some embodiments, the molar percentage of the ionizable cationiclipid is present from about 7.5% to about 20%. In some embodiments, themolar percentage of the ionizable cationic lipid is about 11.9%. In someembodiments, the ionizable cationic lipid is present in a molarpercentage of the lipid nanoparticle composition from about 15% to about30%. In some embodiments, the molar percentage of the ionizable cationiclipid is present from about 15% to about 25%. In some embodiments, themolar percentage of the ionizable cationic lipid is about 20.3%.

In some embodiments, the ionizable cationic lipid comprises an ammoniumgroup which is positively charged at physiological pH and contains atleast two hydrophobic groups. In some embodiments, the ammonium group ispositively charged at a pH from about 6 to about 8. In some embodiments,the ionizable cationic lipid is a dendrimer or dendron. In someembodiments, the ionizable cationic lipid comprises at least two C6-C24alkyl or alkenyl groups. In some embodiments, the ionizable cationiclipid comprises at least two C8-C24 alkyl groups.

In some embodiments, the phospholipid is present in a molar percentageof the lipid nanoparticle composition from about 8% to about 20%. Insome embodiments, the molar percentage of the phospholipid is presentfrom about 10% to about 14%. In some embodiments, the molar percentageof the phospholipid is about 11.9%. In other embodiments, thephospholipid is present in a molar percentage of the lipid nanoparticlecomposition from about 20% to about 23%. In some embodiments, the molarpercentage of the phospholipid is present from about 20% to about 21%.In some embodiments, the molar percentage of the phospholipid is about20.3%. In some embodiments, the phospholipid is further defined as1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or1,2-distearoyl-sn-glycero-3-phosphocholine. In some embodiments, thephospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

In some embodiments, the compositions further comprise a steroid. Insome embodiments, the steroid is present in a molar percentage of thelipid nanoparticle composition from about 39% to about 46%. In someembodiments, the molar percentage of the steroid is present from about40% to about 43%. In some embodiments, the molar percentage of thesteroid is about 40.5%. In other embodiments, the steroid is present ina molar percentage of the lipid nanoparticle composition from about 15%to about 39%. In some embodiments, the molar percentage of the steroidis present from about 20% to about 27.5%. In some embodiments, the molarpercentage of the steroid is about 23.8%. In some embodiments, thesteroid is cholesterol.

In some embodiments, the compositions further comprise a PEGylatedlipid. In some embodiments, the PEGylated lipid is present in a molarpercentage of the lipid nanoparticle composition from about 0.5% toabout 10.0%. In some embodiments, the molar percentage of the PEGylatedlipid is present from about 0.5% to about 5.0%. In other embodiments,the molar percentage of the PEGylated lipid is present from about 2.0%to about 2.8%. In some embodiments, the molar percentage of thePEGylated lipid is about 2.4%. In other embodiments, the PEGylated lipidis present in a molar percentage of the lipid nanoparticle compositionfrom about 3.9% to about 4.6%. In some embodiments, the molar percentageof the PEGylated lipid is present from about 4.0% to about 4.3%. In someembodiments, the molar percentage of the PEGylated lipid is about 4.1%.In some embodiments, the PEGylated lipid comprises a PEG component fromabout 1000 to about 10,000 Daltons. In some embodiments, the PEG lipidis a PEGylated diacylglycerol. In some embodiments, the PEG lipid isfurther defined by the formula:

wherein:

-   -   R₁₂ and R₁₃ are each independently alkyl_((C≤24)),        alkenyl_((C≤24)), or a substituted version of either of these        groups;    -   R_(e) is hydrogen, alkyl_((C≤8)), or substituted alkyl_((C≤8));        and    -   x is 1-250.

In some embodiments, the PEG lipid is dimyristoyl-sn-glycerol or acompound of the formula:

wherein:

-   -   n₁ is 5-250; and n₂ and n₃ are each independently 2-25.

In some embodiments, the compositions comprise cholesterol and DMG-PEG.

In some embodiments, the compositions comprise DOPE. In otherembodiments, the compositions comprise DSPC. In some embodiments, thecompositions further comprise DLin-MC3-DMA. In other embodiments, thecompositions further comprise C12-200. In some embodiments, thecompositions further comprise 3A5-SC8, 3A3-SC8, 4A1-SC8, 4A3-SC8,5A2-SC8 with five tails, or 5A2-SC8 with six tails. In some embodiments,the compositions further comprise 5A2-SC8. In some embodiments, thecompositions further comprise DOTAP. In some embodiments, thecompositions comprise cholesterol, DMG-PEG, DSPC, DLin-MC3-DMA, andDOTAP.

In some embodiments, the therapeutic agent is a small molecule such as asmall molecule selected from an anticancer agents, antifungal agents,psychiatric agents such as analgesics, consciousness level-alteringagents such as anesthetic agents or hypnotics, nonsteroidalanti-inflammatory drugs (NSAIDS), anthelminthics, antiacne agents,antianginal agents, antiarrhythmic agents, anti-asthma agents,antibacterial agents, anti-benign prostate hypertrophy agents,anticoagulants, antidepressants, antidiabetics, antiemetics,antiepileptics, antigout agents, antihypertensive agents,anti-inflammatory agents, antimalarials, antimigraine agents,antimuscarinic agents, antineoplastic agents, antiobesity agents,antiosteoporosis agents, antiparkinsonian agents, antiproliferativeagents, antiprotozoal agents, antithyroid agents, antitussive agent,anti-urinary incontinence agents, antiviral agents, anxiolytic agents,appetite suppressants, beta-blockers, cardiac inotropic agents,chemotherapeutic drugs, cognition enhancers, contraceptives,corticosteroids, Cox-2 inhibitors, diuretics, erectile dysfunctionimprovement agents, expectorants, gastrointestinal agents, histaminereceptor antagonists, immunosuppressants, keratolytics, lipid regulatingagents, leukotriene inhibitors, macrolides, muscle relaxants,neuroleptics, nutritional agents, opioid analgesics, proteaseinhibitors, or sedatives. In other embodiments, the therapeutic agent isa protein. In other embodiments, the therapeutic agent is a nucleic acidsuch as a therapeutic nucleic acid. In some embodiments, the nucleicacid is an siRNA, a miRNA, a pri-miRNA, a messenger RNA (mRNA), aclustered regularly interspaced short palindromic repeats (CRISPR)related nucleic acid, a single guide RNA (sgRNA), a CRISPR-RNA (crRNA),a trans-activating crRNA (tracrRNA), a plasmid DNA (pDNA), a transferRNA (tRNA), an antisense oligonucleotide (ASO), a guide RNA, a doublestranded DNA (dsDNA), a single stranded DNA (ssDNA), a single strandedRNA (ssRNA), and a double stranded RNA (dsRNA). In some embodiments, thecompositions comprise a first nucleic acid and a second nucleic acid. Insome embodiments, the first nucleic acid is a messenger RNA. In someembodiments, the second nucleic acid is a single guide RNA. In someembodiments, the first nucleic acid is a messenger RNA (mRNA) and asingle guide RNA (sgRNA). In some embodiments, the nucleic acid ispresent in a ratio of the lipid nanoparticle composition to the nucleicacid is from about 1:1 to about 1:100. In some embodiments, the ratio isfrom about 1:10 to about 1:60. In some embodiments, the ratio is about1:40.

In some embodiments, the compositions further comprise a protein. Insome embodiments, the protein is a protein associated with translationor transcription. In some embodiments, the protein is associated with aCRISPR process. In some embodiments, the protein is a CRISPR associatedprotein. In some embodiments, the protein is Cas1, Cas1B, Cas2, Cas3,Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2,Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4,Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3,Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modifiedversions thereof. In some embodiments, the protein is Cas9. In someembodiments, the protein and the nucleic acid are present in a molarratio from about 1:1 to about 1:20. In some embodiments, the molar ratiois from about 1:1 to about 1:10. In some embodiments, the molar ratio isfrom about 1:3 to about 1:8.

In some embodiments, the compositions have a negative zeta potential. Insome embodiments, the zeta potential is from −0.25 mV to about −10 mV.In some embodiments, the zeta potential is from about −0.5 mV to about−2 mV. In some embodiments, the compositions comprise both a protein anda nucleic acid. In some embodiments, the compositions comprise Cas9protein and a single guide nucleic acid. In some embodiments, thecomposition comprises Cas9 protein, a single guide nucleic acid, and adonor DNA.

In another aspect, the present disclosure provides pharmaceuticalcompositions comprising:

(A) a composition described herein; and

(B) an excipient.

In some embodiments, the pharmaceutical compositions are formulated foradministration: orally, intraadiposally, intraarterially,intraarticularly, intracranially, intradermally, intralesionally,intramuscularly, intranasally, intraocularly, intrapericardially,intraperitoneally, intrapleurally, intraprostatically, intrarectally,intrathecally, intratracheally, intratumorally, intraumbilically,intravaginally, intravenously, intravesicularlly, intravitreally,liposomally, locally, mucosally, parenterally, rectally,subconjunctival, subcutaneously, sublingually, topically, transbuccally,transdermally, vaginally, in crèmes, in lipid compositions, via acatheter, via a lavage, via continuous infusion, via infusion, viainhalation, via injection, via local delivery, or via localizedperfusion. In some embodiments, the pharmaceutical compositions areformulated for intravenous or intraarterial injection. In someembodiments, the excipient is a pharmaceutically acceptable carrier. Insome embodiments, the pharmaceutically acceptable carrier is a solventor solution. In some embodiments, the pharmaceutical compositions areformulated as a unit dose.

In still another aspect, the present disclosure provides methods ofmodulating the expression of a gene comprising delivering a nucleic acidto a cell, the method comprising contacting the cell with a compositionor a pharmaceutical composition described herein under conditionssufficient to cause uptake of the nucleic acid into the cell.

In some embodiments, the cell is contacted in vitro or ex vivo. In someembodiments, the cell is contacted in vivo. In some embodiments, themodulation of the gene expression is sufficient to treat a disease ordisorder such as cancer.

In still yet another aspect, the present disclosure provides methods oftreating a disease or disorder in a patient comprising administering tothe patient in need thereof a pharmaceutically effective amount of acomposition or a pharmaceutical composition described herein, whereinthe composition or pharmaceutical composition comprises a therapeuticnucleic acid or protein for the disease or disorder.

In some embodiments, the disease or disorder is cancer. In someembodiments, the methods further comprise administering one or moreadditional cancer therapies to the patient. In some embodiments, thecancer therapy is a chemotherapeutic compound, surgery, radiationtherapy, or immunotherapy. In some embodiments, the composition orpharmaceutical composition is administered to the patient once. In otherembodiments, the composition or pharmaceutical composition isadministered to the patient two or more times.

In some embodiments, the patient is a mammal such as a human.

In still other aspects, the present disclosure provides methods ofpreparing a lipid nanoparticle comprising:

-   -   (A) dissolving a permanently cationic lipid, an ionizable        cationic lipid, and a phospholipid in a first solution to form a        lipid solution wherein the lipid solution is formed in an        organic solvent;    -   (B) dissolving a therapeutic agent in a buffer, wherein the        buffer is a buffer with a pH from about 6.8 to about 7.6 to form        a buffered therapeutic agent solution; and    -   (C) admixing the lipid solution to the buffered therapeutic        agent solution to form a lipid nanoparticle.

In some embodiments, the organic solvent is a C1-C4 alcoholic solventsuch as ethanol. In some embodiments, the buffer is an aqueous PBSbuffer. In some embodiments, the methods have an encapsulatingefficiency of greater than 80%.

In yet another aspect, the present disclosure provides compositionscomprising:

(A) a therapeutic agent;

(B) a lipid nanoparticle composition comprising:

-   -   (1) an ionizable cationic lipid;    -   (2) a phospholipid; and    -   (3) a selective organ targeting compound;        wherein the organ targeting ligand causes the preferential        delivery of the composition to an organ other than the liver.

In still yet another aspect, the present disclosure providescompositions comprising:

(A) a therapeutic agent;

(B) a lipid nanoparticle composition comprising:

-   -   (1) an ionizable cationic lipid;    -   (2) a phospholipid;    -   (3) a selective organ targeting compound;    -   (4) a steroid; and    -   (5) a PEG lipid;        wherein the organ targeting ligand causes the preferential        delivery of the composition to an organ other than the liver.

In still another aspect, the present disclosure provides compositionscomprising a therapeutic agent and a lipid nanoparticle compositioncomprising:

(A) an ionizable cationic lipid;

(B) a phospholipid; and

(C) a selective organ targeting compound;

wherein the composition has an apparent pK_(a) from about 8 to about 13and the composition primarily delivers the nucleic acid to the lungs.

In another aspect, the present disclosure provides compositionscomprising a therapeutic agent and a lipid nanoparticle compositioncomprising:

(A) an ionizable cationic lipid;

(B) a phospholipid;

(C) a selective organ targeting compound;

(D) a steroid; and

(E) a PEG lipid;

wherein the composition has an apparent pK_(a) from about 8 to about 13and the composition primarily delivers the nucleic acid to the lungs.

In still another aspect, the present disclosure provides compositionscomprising a therapeutic agent and a lipid nanoparticle compositioncomprising:

(A) an ionizable cationic lipid;

(B) a phospholipid; and

(C) a selective organ targeting compound;

wherein the composition has an apparent pK_(a) from about 3 to about 6and the composition primarily delivers the nucleic acid to the spleen.

In still yet another aspect, the present disclosure providescompositions comprising a therapeutic agent and a lipid nanoparticlecomposition comprising:

(A) a steroid;

(B) an ionizable cationic lipid;

(C) a phospholipid;

(D) a PEG lipid; and

(E) a selective organ targeting compound;

wherein the composition has an apparent pK_(a) from about 3 to about 6and the composition primarily delivers the nucleic acid to the spleen.

In another aspect, the present disclosure provides compositionscomprising a therapeutic agent and a lipid nanoparticle compositioncomprising:

(A) an ionizable cationic lipid;

(B) a phospholipid; and

(C) a C₆-C₂₄ diacyl phosphotidylcholine;

wherein the composition primarily delivers the nucleic acid to the lymphnodes.

In still another aspect, the present disclosure provides compositionscomprising a therapeutic agent and a lipid nanoparticle compositioncomprising:

(A) a steroid;

(B) an ionizable cationic lipid;

(C) a phospholipid;

(D) a PEG lipid; and

(E) a C₆-C₂₄ diacyl phosphotidylcholine;

wherein the composition primarily delivers the nucleic acid to the lymphnodes.

In still yet another aspect, the present disclosure providescompositions comprising a therapeutic agent and a lipid nanoparticlecomposition comprising:

(A) an ionizable cationic lipid;

(B) a phospholipid; and

(C) a selective organ targeting compound;

wherein the surface of the composition interacts with vitronectin andthe composition primarily delivers the nucleic acid to the lungs.

In yet another aspect, the present disclosure provides compositionscomprising a therapeutic agent and a lipid nanoparticle compositioncomprising:

(A) an ionizable cationic lipid;

(B) a phospholipid;

(C) a selective organ targeting compound;

(D) a steroid; and

(E) a PEG lipid;

wherein the surface of the composition interacts with vitronectin andthe composition primarily delivers the nucleic acid to the lungs.

In still yet another aspect, the present disclosure providescompositions composition comprising a therapeutic agent and a lipidnanoparticle composition comprising:

(A) an ionizable cationic lipid;

(B) a phospholipid; and

(C) a selective organ targeting compound;

wherein the surface of the composition interacts with Apo H and thecomposition primarily delivers the nucleic acid to the spleen.

In another aspect, the present disclosure provides compositionscomposition comprising a therapeutic agent and a lipid nanoparticlecomposition comprising:

(A) a steroid;

(B) an ionizable cationic lipid;

(C) a phospholipid;

(D) a PEG lipid; and

(E) a selective organ targeting compound;

wherein the surface of the composition interacts with Apo H and thecomposition primarily delivers the nucleic acid to the spleen.

In still yet another aspect, the present disclosure providescompositions comprising a therapeutic agent and a lipid nanoparticlecomposition wherein a targeting protein present in a protein corona atthe surface of the composition binds to a target protein substantiallypresent in the target organ, wherein the target organ is not the liver.

In some embodiments, the targeting protein is selected from vitronectinor β2-glycoprotein I (Apo H). In some embodiments, the targeting proteinis vitronectin and the target organ is the lungs. In other embodiments,the targeting protein is Apo H and the target organ is the spleen.

In some embodiments, the lipid nanoparticle composition furthercomprises a selective organ targeting compound which modifies thebinding of the proteins on the protein corona. In some embodiments, theselective organ targeting compound is further selected from a sugar, alipid, a small molecule therapeutic agent, a vitamin, or a protein. Insome embodiments, the selective organ targeting compound is a lipid suchas a permanently cationic lipid, a permanently anionic lipid, or aphosphotidylcholine. In some embodiments, the lipid nanoparticlecomposition further comprises an ionizable cationic lipid. In someembodiments, the lipid nanoparticle composition further comprises aphospholipid. In some embodiments, the lipid nanoparticle compositionfurther comprises a steroid. In some embodiments, the lipid nanoparticlecomposition further comprises a PEG lipid.

In another aspect, the present disclosure provides compositionscomprising a therapeutic agent and a lipid nanoparticle composition,wherein the lipid nanoparticle composition comprises a selective organtargeting compound and the selective organ targeting compound results ina lipid nanoparticle composition with an apparent pK_(a) from about 3 toabout 6.

In still another aspect, the present disclosure provides compositionscomprising a therapeutic agent and a lipid nanoparticle composition,wherein the lipid nanoparticle composition comprises a selective organtargeting compound and the selective organ targeting compound results ina lipid nanoparticle composition with an apparent pK_(a) from about 8 toabout 13.

As used herein, “essentially free,” in terms of a specified component,is used herein to mean that none of the specified component has beenpurposefully formulated into a composition and/or is present only as acontaminant or in trace amounts. The total amount of the specifiedcomponent resulting from any unintended contamination of a compositionis preferably below 0.01%. Most preferred is a composition in which noamount of the specified component can be detected with standardanalytical methods.

As used herein in the specification and claims, “a” or “an” may mean oneor more. As used herein in the specification and claims, when used inconjunction with the word “comprising”, the words “a” or “an” may meanone or more than one. As used herein, in the specification and claim,“another” or “a further” may mean at least a second or more.

As used herein in the specification and claims, the term “about” is usedto indicate that a value includes the inherent variation of error forthe device, the method being employed to determine the value, or thevariation that exists among the study subjects.

Other objects, features and advantages of the present disclosure willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating certain embodiments of the disclosure, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The disclosure may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIGS. 1A-C: DOTAP mDLNP formulations mediated excellent mRNA deliveryefficacy at low doses after IV injection and demonstrated tissuespecific delivery features with given percentage of DOTAP. (FIG. 1A)Schematic illustration of DOTAP mDLNP formation and the structure ofDOTAP. The molar ratio of 5A2-SC8/DOPE/Chol/DMG-PEG were fixed as15/15/30/3 (named mDLNP). The DOTAP ratios only were then adjusted from0 to 1200 to produce a series of DOTAP mDLNP formulations and namedDOTAPY, where Y represented the percentage of DOTAP in total lipids.(FIG. 1B) Ex vivo images of luciferase in major organs at 6 h post IVinjection with the dose of 0.1 mg/kg Luc mRNA (n=2). With increasingmolar percentage of DOTAP, luciferase protein expression moved fromliver to spleen, then to lung. (FIG. 1C) Quantification datademonstrated that DOTAP percentage is a factor for tissue specificdelivery, mDLNP (0%) was the best for liver, DOTAP10-15 (similar betweenthem) were the best for spleen, and DOTAP50 was the best for lung.Because luciferase expression was detected only in liver, spleen andlung after IV injection, the relative expression in each organ wascalculated. Clearly, the more DOTAP (permanently cationic lipid)percentage in formulation, the less luminescence in liver and close to 0when >70%. However, the more DOTAP percentage, the more luminescence inlung and close to 100% when >70%. The DOTAP5-20 showed higherpercentages in spleen and DOTAP10 looked the highest.

FIGS. 2A1-2F2: The structures of lipids decide mRNA expression profileafter IV injection. Generally, quaternary lipids changed mRNA deliveryfrom liver to spleen, then to lung with increasing percentages, andzwitterionic lipids helped to deliver mRNA into spleen at higherpercentages. But tertiary amine lipids could not change mRNA expressionorgan, instead improved delivery efficacy in liver. To further confirmthe delivery trend of quaternary lipids mDLNPs, two more quaternarylipids, DDAB and EPC, were selected to perform mRNA delivery in vivo,with the same modified strategy as was done with DOTAP. (FIG. 2A1, FIG.2B1) DDAB and EPC exhibit large structural differences between them andwith DOTAP, including on three levels of comparison: the length ofhydrophobic tail, saturated and unsaturated bond, and chemical structureof head group. Formulations with 5%, 15%, 40% and 50% of quaternarylipid were formed to detect size distribution and in vivo evaluation(0.1 mg/kg, 6 h, n=2). (FIG. 2A2, FIG. 2B2) Like DOTAP mDLNPs, DDAB andEPC also presented similar mRNA delivery profile. Lower cationic percent(5%) delivered mRNA into liver and spleen, then to spleen more whenincreased to 15%. Once increased to 40%, little mRNA expression wasobserved in liver and spleen, but lung exhibited high luciferase signaland then decreased at 50%. These results are very similar with DOTAPmDLNP, which suggested that functionalized mDLNP by quaternary lipidsare universal and generalizable strategy for tissue targeting mRNAdelivery. Then representative zwitterionic lipids, DSPC and DOCPe, wereused to evaluate the mRNA delivery in vivo just like DOTAP strategy(FIG. 2C1, FIG. 2D1). The structures of DSPC and DOCPe lipids are in thegeneral class of zwitterionic lipids. Size distributions of DSPC andDOCPe mDLNP formulations were tested by DLS before IV injection. Herein,DSPC and DOCPe showed two levels comparison in structures: saturatedversus unsaturated hydrophobic tails, and charge position versus in headgroups. (FIG. 2C2, FIG. 2D2) Interestingly, a similar mRNA expressionprofile like quaternary lipid formulations was not observed. Instead,both DSPC and DOCPe improved mRNA delivery into spleen within givenrange (less than 80% in DSPC and less than 50% in DOCPe), never saw anysignals in lung whatever percentages (0.1 mg/kg, 6 h, n=2). Inspired bythese results, ionizable tertiary amine lipids, DODAP and C12-200, werefurther tested with the same strategy. DODAP has the same structure withDOTAP except for the head groups (quaternary versus tertiary amine),C12-200 is an effective lipidoid used for siRNA or mRNA delivery, whichhas a completely different structure with DODAP. (FIG. 2E1, FIG. 2F1)Similarly, the size distributions of both modified mDLNPs were stillgood at certain percentages (less than 80%). (FIG. 2E2, FIG. 2F2)Surprisingly, DODAP and C12-200 could not change mRNA expression profile(different effect compared to quaternary or zwitterionic lipids).Instead, DODAP and C12-200 increased delivery of mRNA to the liver.Supporting that, DODAP20 and C12-200 showed much better deliveryefficacy than original mDLNP formulation (0.1 mg/kg, 6 h, n=2). Withincreasing percentages of DODAP or C12-200 (50% or 80%), luciferasesignal decreased a lot, but liver still was the main organ rather thanspleen or lung.

FIGS. 3A-C: To determine why various lipids may induce huge differencesfor mRNA expression in organs, distribution assay and pKa detection wasthen performed. Both biodistribution and pKa played roles for mRNAexpression profiles in organs. (FIG. 3A) Organs distribution ofCy5.5-Luc mRNA formulations delivered by three kinds of modified mDLNP,DOTAP (quaternary lipid), DSPC (zwitterionic lipid) and DODAP (tertiaryamine lipid). C57BL/6 mice were IV injected at the doses of 0.5 mg/kgand imaged at 6 h post injection (n=2). DOTAP changed the organdistribution of mRNA, compared with the original mDLNP formulation (noDOTAP), both DOTAP10 and DOTAP50 could deliver mRNA into lung, andDOTAP50 increased more, which may partly explain why DOTAP formulationmediated mRNA expression in lung at higher percentages. However, bothDSPC and DODAP could not change mRNA distribution much even at 80%(DSPC) or 50% (DODAP) percentages. It was also noticed that mRNA wasretained in liver for DOTAP50 and DSPC80, as showed in FIGS. 1 and 2,the former one was lung targeting NP and latter one was spleen targetingNP. Therefore, distribution was not the only factor to explain themechanism. (FIG. 3B) The pKa of all tested and efficacious formulationswere then measured, including original mDLNP, DOTAP, DDAB, EPC, DSCP,DOCPe, DODAP and C12-200 modified formulations. (FIG. 3C) Finallyrelationship between pKa and tissue specific mRNA delivery was plottedbased on the defined rules. Here, 8 rules to score were designed asshowed in the table. Obviously, all liver targeted formulations had anarrow pKa (˜6-7), no significant range for spleen targeted formulation,but lung targeted delivery required high pKa (>9.25).

FIGS. 4A-4C: Liver and lung gene editing were achieved both in Td-Tomatoand C57BL/6 mice. (FIG. 4A) Schematic illustration shows thatco-delivery of Cas9 mRNA and sgTom1 activates td-tomato expression inTd-Tomato mouse. (FIG. 4B) Td-tomato expressions were induced in liverand lung when treated by mDLNP and DOTAP50 formulations, respectively.The mice were IV injected by mDLNP and DOTAP50 formulations forco-delivery of IVT Cas9 mRNA and modified sgTom1 (4/1, wt/wt) at thetotal doses of 2.5 mg/kg (50 μg each), then fluorescence of main organswas detected at day 10 after treatment. (FIG. 4C) T7E1 assay showed thattissue specific feature was further confirmed with in vivo PTEN editing.C57 BL6 mice were IV injected with mDLNP, DODAP20 or DOTAP50 to reachtissue specific gene editing, total dose was 2.5 mg/kg (50 μg each),weight ratio of IVT Cas9 mRNA to modified sgPTEN was 4/1, and detectiontime was day 10 after treatment.

FIGS. 5A-C: Characterization of DOTAP mDLNP formulations (n=3). Size,PDI (FIG. 5A) and zeta-potential (FIG. 5B) were detected by DynamicLight Scattering (DLS). (FIG. 5C) Encapsulation efficiency (EE %) wastested by Ribogreen RNA assay.

FIGS. 6A-C: DOTAP formulations showed excellent mRNA delivery efficiencyand exhibited a delivery potential for these cargos that were notwell-tolerated with ethanol or acidic buffer, e.g. proteins. (FIG. 6A)DOTAP mDLNP mediated high Luc mRNA expression in Huh-7 cells and A549cells and revealed that DOTAP percentages of 5%-50% were better for mRNAdelivery and 10% was the best. Luc mRNA expression and cell viabilitywere tested at 24 h post transfection with the dose of 50 ng/well mRNA(n=4). Here, DOTAP mDLNPs were formed in PBS rather than citric buffer(10 mM, pH 4.0). (FIGS. 6B & 6C) Decreased volume percentage of ethanoldid not affect the characterization and mRNA delivery potency. To testthe influence of ethanol for mRNA delivery, DOTAP25 was selected as amodel and formed four formulations with various volume ratios of ethanolto PBS (1:3, 1:5, 1:7.5 and 1:10). All four formulations showed similarEE, size and PDI (FIG. 6B), and exhibited equality mRNA delivery potency(FIG. 6C) in FaDu cells (50 ng/well mRNA, 24 h, n=4). Therefore, thisformulation was optimized using 1×PBS (pH 7.4) to instead of acidicbuffer (10 mM pH 4.0) and dramatically decreased ethanol percentage,which provide a possibility that DOTAP formulations deliver those cargosthat are not well-tolerated in high ethanol concentrations or acidicbuffer, e.g. proteins.

FIG. 7: The quantification data of biodistribution in main organs. C57BL6 mice were IV injected by various Cy5.5-Luc mRNA formulations at thedoses of 0.5 mg/kg (n=2). Heart, lung, liver, spleen and kidney wereisolated, imaged and quantified after 6 h.

FIGS. 8A-8C: There were no big differences for size distribution and LucmRNA delivery efficacy for DOTAP10 formulation formed by PBS or citricbuffer, but not applied in DSPC50 and DODAP50. To test the buffereffects on mRNA delivery efficacy in vivo, DOTAP10 (quaternary lipid),DSPC50 (zwitterionic lipid) and DODAP50 (tertiary amine lipid) werechosen. C57 BL6 mice were IV injected by each Luc mRNA formulation atthe dose of 0.1 mg/kg, after 6 h, main organs were isolated and imaged(n=2). (FIG. 8A) Both size and delivery efficacy were not changed toomuch between PBS formed and citric buffer (10 mM, pH 4.0) formedDOTAP10. (FIGS. 8B & 8C) But citric buffer formed DSPC50 and DODAP50dramatically improved mRNA delivery effects, although no big differenceon size distributions. DOTAP (or another permanently cationic lipid) maybe added for LNP formation at neutral pH (e.g. 7.4 PBS buffer).

FIG. 9: Western blot result of IVT Cas9 mRNA quality test delivered bymDLNP. To achieve tissue specific gene editing, Cas9 mRNA and sgRNA weredesigned to be co-delivered. Firstly, Cas9 mRNA was made by IVT andanalyzed by western blot for quality test. In this assay, both Cas9 pDNAdelivered by Lipofectamine 2000 and commercial Cas9 mRNA (TriLink).mDLNP were positive controls, and mCherry mDLNP was negative control.293T cells were seeded in 12-well plate the day before transfection, andcells were treated for 24 h by each condition before performing westernblot. IVT Cas9 mRNA worked much better than commercial mRNA, therefore,IVT Cas9 mRNA was used to perform gene editing in vivo.

FIGS. 10A & 10B: sgRNAs screening and weight ratio (Cas9 mRNA/sgRNA)optimization in Td-Tomato mice. To reach maximum gene editing inTd-Tomato mice, sgRNA sequences were screened and optimized weight ratioof Cas9 mRNA to sgRNA. (FIG. 10A) Size distributions of Cas9/sgTom1,Cas9/sgTom2 and Cas9/sgLoxP mDLNP formulations, and td-tomato expressionin liver. Mice were IV injected by three mDLNP at the total dose(Cas9/sgRNA, 4/1, wt/wt) of 3 mg/kg, organ were imaged at day 7. Itlooked that sgTom1 is the lead among three candidates. (FIG. 10B) SgTom1was selected and further tested by different weight ratios (Cas9/sgRNA)of 2/1, 4/1 and 6/1 for liver gene editing. mDLNP was used for IVinjection with the dose of 3 mg/kg, and td-tomato expression wasdetected in day 7. In this example, 4/1 worked better than 2/1 and 6/1.

FIGS. 11A-11G: Characterization of Cas9/sgRNA complex and DOTNP lipidnanoparticles after encapsulating Cas9/sgRNA complex. Size (FIG. 11A)and Zeta potential (FIG. 11B) of Cas9/sgLUC complex(mol/mol=1/1) in PBS(pH7.4) and in Citrate Buffer (pH4.2). Size of Cas9/sgLUC complexprepared in citrate buffer is very big (larger than 100 nm) and the zetapotential is positively charged, so it is impossible to be encapsulatedby lipid nanoparticles. However, the size of Cas9/sgLUC complex preparedin PBS is compact (less than 20 nm) and has negative charges, so it isable to be encapsulated by lipid nanoparticles. Size (FIG. 11C) and Zetapotential (FIG. 11D) of Cas9/sgLUC complex prepared with differentCas9/sgRNA mole ratios (1/1, 1/3, and 1/5). Compared with Cas9/sgLUCcomplex (1/1, mol/mol), higher mole ratios (1/3 and 1/5, mol/mol) showedsmaller size and more negative charges, which are beneficial for lipidnanoparticle encapsulation. Size (FIG. 11E) and Zeta potential (FIG.11F) of DOTNP10 lipid nanoparticles encapsulating Cas9/sgLUC complex(named DOTNP10-L) when preparing with different mole ratios (1/1, /3,1/5). (FIG. 11G) TEM images of DOTNP10-L (1/3, mol/mol). DOTAP lipidnanoparticle consists of five components, including 5A2-SC8,Cholesterol, DOPE, DMG-PEG and DOTAP. The mole ratio of 5A2-SC8,Cholesterol, DOPE, and DMG-PEG is fixed (15:15:30:5, mol/mol) and DOTNPXmeans DOTNP with different mole percentage of DOTAP. Here, differentsgRNA including sgLUC, sgGFP, sgTOM, sgPTEN etc. were used. Todistinguish them, the first letter of each gene was added at the end ofDOTNP. For example, DOTNP10-L means DOTNP10 lipid nanoparticlesencapsulating Cas9/sgLUC complex; DOTNP10-G means DOTNP10 lipidnanoparticles encapsulating Cas9/sgGFP complex.

FIGS. 12A-12F: DOTNP lipid nanoparticles were able to deliver Cas9/sgRNAcomplex into nucleus and exhibited efficient gene editing in vitro.(FIG. 12A) Confocal images of Hela-Luc cells after incubated withDOTNP10 encapsulating Cas9-EGFP/sgLUC complexes (1/3, mol/mol) for 1 h,3 h, 6 h, and 24 h (9 nM of sgRNA was used). Green: EGFP fused Cas9protein; Blue: nuclei stained with Hoechst 33342. Red arrows indicatedthe process of DOTNP10 entering into nucleus. (FIG. 12B) Indelspercentages at LUC locus after incubated with DOTNP10-L of differentmole ratios for 3 days analyzed by TIDE Sequencing (24 nM of sgRNA wasused). DOTNP10 lipid nanoparticles encapsulating Cas9/sgGFP (DOTNP10-G)was used as negative control. Here, two commercial Cas9 proteins(GeneArt Cas9 and Truecut Cas9) were used. (FIG. 12C) T7EI cleavageassay of Hela-Luc cells incubated with different formulations (24 nM ofsgRNA was used). 1. 100 bp DNA ladder; 2. PBS; 3. DOTNP10-G (1/3); 4.DOTNP10-L (1/1); 5. DOTNP10-L (1/3); 6. DOTNP10-L (1/5); 7.DOTNP10-L(1/3) prepared in Citrate buffer. Two commercial Cas9 proteins (GeneArtCas9 and Truecut Cas9) were used. Among them, mole ratio at 1/3 showedthe best gene editing when using Truecut Cas9 protein. (FIG. 12D)Fluorescence microscopy images of SKOV3-GFP cells incubated withDOTNP10-L and DOTNP10-G (24 nM of sgRNA was used). Here, DOTNP10-L wasused as negative control. (FIG. 12E) Flow cytometry analysis ofSKOV3-GFP cells incubated with DOTNP10-L and DOTNP10-G. (FIG. 12F) Meanfluorescence intensity of SKOV3-GFP cells incubated with DOTNP10-L andDOTNP10-G by flow cytometry.

FIGS. 13A & 13B: DOTNPs showed tissue-specific gene editing in vivo.(FIG. 13A) Ex vivo images of tdTomato fluorescence in major organs at 7days post IV injection of different formulations (1.5 mg/kg of sgRNA permouse). DOTNP5-T means DOTNP5 lipid nanoparticles encapsulatingCas9/sgTom complex; DOTNP10-T means DOTNP10 lipid nanoparticlesencapsulating Cas9/sgTom complex; DOTNP50-T means DOTNP50 lipidnanoparticles encapsulating Cas9/sgTom complex. tdTomato fluorescencewas only observed in liver in DOTNP5-T treated group; In DOTNP10-Tgroup, slight fluorescence was seen in lung and if further increasingdose of DOTAP to 50% (DOTNP50-T), most of tdTomato fluorescence wasobserved in lung. (FIG. 13B) T7EI cleavage assay of liver and lungorgans after incubating with DOTNP5-P (DOTNP5 lipid nanoparticlesencapsulating Cas9/sgPTEN complex), DOTNP10-P (DOTNP10 lipidnanoparticles encapsulating Cas9/sgPTEN complex) and DOTNP50-P (DOTNP50lipid nanoparticles encapsulating Cas9/sgPTEN complex) (2 mg/kg of sgRNAper mouse). The results are consistent with that obtained by ex vivoimaging. Gene editing was only detected in liver after treated withDOTNP5-P; gene editing was obtained both in liver and in lung whenincubated with DOTNP10-P; while in DOTNP50-P treatment group, most ofgene editing was observed in lung.

FIGS. 14A & 14B show the details of MC3 LNPs and DOTAP modified MC3formulations, including (FIG. 14A) structures of each component, (FIG.14B) molar ratios, weight ratios of total lipid to mRNA, sizes and PDI.

FIGS. 15A & 15B show DLin-MC3-DMA (FIG. 15A) and C12-200 (FIG. 15B) wereselected and evaluated with the DOTAP strategy at the doses of 0.1 mg/kg(6 h, n=2). With increasing DOTAP percentages from 0 to 50%, both MC3and C12-200 based LNPs showed identical mRNA expression profiles likemDLNP where luciferase signal moved from liver to spleen and finally tolung.

FIGS. 16A & 16B show the details of C12-200 LNPs and DOTAP modifiedC12-200 formulations, including (FIG. 16A) structures of each component,(FIG. 16B) molar ratios, weight ratios of total lipid to mRNA, sizes andPDI.

FIGS. 17A & 17B show (FIG. 17A) Further optimization of mDLNP. As thekey lipid in mDLNP, 5A2-SC8 was used as the “fifth” lipid to modifymDLNP to form four formulations with extra percentage of 10% to 30%.(FIG. 17B) Ex vivo luciferase images and quantified data showed thatmRNA delivery potency was dramatically improved with extra 15% to 25% of5A2-SC8, and 20% had the highest signal (0.05 mg/kg, 6 h, n=2).

FIG. 18 shows the structures of 5A2-SC8, DOPE, Cholesterol and DMG-PEG.mDLNP is an effective and safe mRNA delivery carrier for liver targetingtherapeutics developed by previous work, which is consisted with5A2-SC8, DOPE, Cholesterol and DME-PEG at the molar ratio of 15/15/30/3.

FIGS. 19A-19G show selective ORgan Targeting (SORT) allows lipidnanoparticles (LNPs) to be systematically and predictably engineered toaccurately edit cells in specific organs. (19A) Addition of asupplemental component (termed a SORT lipid) to traditional LNPssystematically alters the in vivo delivery profile and mediates tissuespecific delivery as a function of the percentage and biophysicalproperty of the SORT lipid. This universal methodology successfullyredirected multiple classes of nanoparticles. Shown here arebioluminescence images of mice injected intravenously with 0.1 mg/kgLuciferase mRNA inside of lung- and spleen-specific DLin-MC3-DMA LNPs(Onpattro SNALPs) and liver enhanced 5A2-SC8 degradable dendrimer-basedLNPs (DLNPs). Inclusion of SORT lipids into 4-component 5A2-SC8,DLin-MC3-DMA, and C12-200 LNPs created 5-component SORT LNPs. (19B)5A2-SC8 SORT LNPs were formulated with a molar ratio of5A2-SC8/DOPE/Chol/DMG-PEG/SORT Lipid=15/15/30/3/X (mol/mol), where X wasadjusted from 0 to 1200 to make series of LNPs with 0% to 100% SORTlipid (fraction of total lipids). Here, inclusion of a permanentlycationic lipid (DOTAP) systematically shifted luciferase proteinexpression from the liver to spleen to lung as a function of DOTAPpercentage (0.1 mg/kg Luc mRNA, 6 h). (19C) Quantification datademonstrated that SORT lipid percentage is a factor for tissue specificdelivery; 0% (mDLNPs) was optimal for liver; 5-15% was optimal forspleen; and 50% was optimal for lungs. (19D) Relative luciferaseexpression in each organ demonstrated that fractional expression couldbe predictable tuned. (19E) Inclusion of an anionic SORT lipid enabledselective mRNA delivery to the spleen. Luciferase expression wasobserved only in spleen when introducing 18PA lipid into mDLNPs up to40% (0.1 mg/kg Luc mRNA, 6 h). (19F) Ex vivo images of luminescence inmajor organs at 6 h post IV injection of DLin-MC3-DMA SORT LNPs with thedose of 0.1 mg/kg Luc mRNA. With increasing molar percentage of DOTAP,luciferase expression moved from liver to lung. 18PA mediated exclusivedelivery of Luc mRNA to the spleen. The same trend was observed formodified C12-200 LNPs (0.1 mg/kg, 6 h). (19G) Details of selected SORTlipid formulations.

FIGS. 20A-20C show (20A) Details of DOTAP and 18PA SORT LNPs, includingmolar ratios, molar percentages, weight ratios of total lipid to mRNA,sizes, PDI and zeta potentials. (20B) LNPs were formulated using amodified ethanol dilution method. SORT lipids are included in theethanol phase and sgRNA/mRNA are encapsulated during LNP formation.(20C) The chemical structures of lipids used in standard mDLNP andDOTAP/18PA SORT formulations are shown. For the development of SORT, adegradable dendrimer-based ionizable cationic lipid named 5A2-SC8 wasthe focused of the LNPs that can deliver siRNAs/miRNAs to extendsurvival in a genetically engineered mouse model of MYC-driven livercancer (Zhou et al., 2016; Zhang et al., 2018a; Zhang et al., 2018b) andtoggle polyploidy in the liver. An LNP molar composition that wasoptimized for mRNA delivery was focused to the liver named mDLNPs (Chenget al., 2018). This liver-targeted base mRNA formulation of5A2-SC8/DOPE/Cholesterol/DMG-PEG2000=15/15/30/3 (mol) were prepared andsupplemented with SORT lipids to prepare SORT LNPs (details in 20A). Forthe sake of further clarity, the traditional 4-component LNPs arecomposed of ionizable cationic lipids (herein defined as containing anamino group with pK_(a)<8), zwitterionic phospholipids (defined as alipid bearing equal number of positive and negative charges),cholesterol, and poly(ethylene glycol) (PEG) lipids (most commonly,PEG2000-DMG). SORT LNPs include a 5^(th) lipid, such as a permanentlycationic lipid (defined as positively charged without pK_(a) orpK_(a)>8) or a permanently anionic lipid (defined as negativelycharged).

FIGS. 21A & 21B show In vitro Luciferase (Luc) mRNA delivery results forDOTAP-modified SORT mDLNPs in (FIG. 21A) Huh-7 liver cells and (FIG.21B) A549 lung cells as a function of the incorporated DOTAP percentage.Luc mRNA delivery results showed that LNPs with DOTAP percentages of5%-50% delivered the most mRNA in both Huh-7 liver cells and A549 lungcells. SORT LNPs with 10% DOTAP were much more efficacious in vitro thanthe previously reported base mDLNP. No appreciable cytotoxicity wasobserved for any formulation and all were uniform (low PDI) withdiameters ranging from 90 nm to 150 nm (FIG. 20). Measurements ofsurface charge revealed that DOTAP was encapsulated inside together withmRNA and not on the LNP surface as the zeta potentials were close to 0when DOTAP was less than 60%. The surface charge became positive only atpercentages above 65% (FIG. 20), revealing that PEG lipid-coated SORTLNPs with selective tissue tropism could be discovered that possess anear neutral surface charge, which is an attribute for clinicaltranslation. Cells were seeded into 96-well plates at a density of 1×10⁴cells per well the day before transfection. Luc mRNA expression and cellviability were measured at 24 h post treatment with the dose of 50ng/well Luc mRNA (n=4).

FIGS. 22A-22C show chemical structures of lipids used in (22A)DLin-MC3-DMA SNALPS (Jayaraman et al., 2012) and (22B) C12-200 LLNPs(Love et al., 2010) are shown. Liver-targeted base mRNA formulations ofDLin-MC3-DMA/DSPC/Cholesterol/DMG-PEG2000=50/10/38.5/1.5 (mol) andC12-200/DOPE/Cholesterol/DMG-PEG2000=35/16/46.5/2.5 (mol) were preparedand later supplemented with SORT lipids to prepare SORT LNPs. (22C)Table and results of additional SORT formulations using DLin-MC3-DMA andC12-200. The weight ratio of total lipids/mRNA was 20/1 (wt/wt) for allDLin-MC3-DMA and C12-200 LNPs.

FIGS. 23A-23C show SORT relies on general biophysical properties and notexact chemical structures. (23A) SORT lipids could be divided intospecific groups with defined biophysical properties. Permanentlycationic SORT lipids (DDAB, EPC, and DOTAP) all resulted in the samemRNA delivery profile (liver to spleen to lung based on SORT lipidpercentage) (0.1 mg/kg Luc mRNA, 6 h). (23B) Anionic SORT lipids (14PA,18BMP, 18PA) all resulted in the same mRNA delivery profile (exclusivelyspleen based on SORT lipid percentage). (23C) Ionizable cationic SORTlipids with tertiary amino groups (DODAP, C12-200) enhanced liverdelivery without any luciferase expression in the lungs (0.1 mg/kg LucmRNA, 6 h).

FIGS. 24A & 24B show SORT was further applied to utilize ionizablecationic lipids as SORT lipids to further enhance mDLNP liver delivery.(24A) Schematic illustration of SORT. (24B) 5A2-SC8 was used as a SORTlipid, supplementing base the mRNA mDLNP formulation(5A2-SC8/DOPE/Cholesterol/DMG-PEG2000=15/15/30/3 (mol)) with additional5A2-SC8 using the SORT method. Ex vivo luciferase images and quantifieddata showed that mRNA delivery potency was dramatically improved when anextra 15%-25% SORT lipid was added. Maximal expression was produced with20% incorporation (0.05 mg/kg, 6 h, n=2). SORT thus allowed developmentof a 2^(nd) generation mDLNP with increased efficacy.

FIGS. 25A & 25B show the effects of zwitterionic SORT lipids wereevaluated. Inclusion of zwitterionic SORT lipids into liver-targetedmDLNPs altered expression from the liver to the spleen with increasingincorporation of the SORT lipid. 80% DSPC and 50% DOCPe SORT LNPsdelivered mRNA exclusively to the spleen after IV injection. (25A)Schematic illustration of SORT method. (25B) Ex vivo images ofluminescence in major organs at 6 h post IV injection. DSPC and DOCPe,zwitterionic lipids with different structures, improved Luc mRNAdelivery into spleen with increased percentages (0.1 mg/kg, 6 h, n=2).

FIGS. 26A & 26B SORT was evaluated as a potential strategy to “activate”inactive LNP formulations. (26A) Schematic illustration of supplementingan inactive C1 formulation with a SORT lipid to test if SORT can endowactivity. (26B) The detailed information of C1 LNPs (inactive LNPs) andDOTAP (or DODAP) C1 SORT LNPs, including lipid molar ratios, molarpercentages, weight ratios of total lipids to mRNA, sizes, and PDI. C1LNPs were prepared in a way that allowed for mRNA encapsulation andfavorable biophysical properties (uniform <200 nm size). However, noprotein expression at all resulted following IV injection of C1 LNPs.Thus, it was asked if SORT could “activate” dead LNPs. DODAP and DOTAPSORT lipids were evaluated. DODAP@C1 LNPs delivered mRNA into spleen andliver, and DOTAP@C1 LNPs delivered mRNA into lung and spleen (0.1 mg/kg,6 h, n=2). Therefore, SORT can activate dead LNPs and provide tissueselectivity.

FIGS. 27A & 27B show SORT altered LNP biodistribution and revealed acorrelation between relative apparent pKa and organ specificity. (27A)Fluorescent Cy5-labeled mRNA was employed to track biodistribution ofSORT LNPs. Inclusion of DOTAP as a SORT lipid increased mRNAaccumulation in the lungs, partially explaining the ability to deliverRNA to mouse lungs. 18PA increased uptake into the spleen. DODAPslightly increased liver and decreased spleen accumulation (0.5 mg/kg, 6h). Note that this data describes SORT LNP location, not the ability toproductively delivery mRNA intracellularly. (27B) The relative apparentpKas of all 67 effective mRNA formulations were measured by the TNSassay and plotted versus in vivo delivery efficacy in different organs(functional delivery by mRNA translated to protein). All liver targetedformulations had a narrow pKa (6-7), as expected. Surprisingly, high pKa(>9) was required for lung-targeted delivery and lower pKa (<6) aidedspleen delivery. Note that all SORT LNPs contain ionizable cationiclipids (for endosomal escape) along with a mixture of other charged anduncharged lipids (that collectively mediate tissue tropism).

FIGS. 28A & 28B show Cy5-labeled mRNA was employed to trackbiodistribution of SORT LNPs. Organ distributions of DSPC mDLNPs afterIV injection. (28A) Schematic illustration of SORT. (28B) Cy5fluorescence and quantified data of major organs treated by DSPC mDLNPs(0.5 mg/kg, 6 h, n=2).

FIG. 29 shows a modified TNS assay was used to measure global/apparentpKa of mRNA formulations. 67 NPs successful formulations (high in vivoefficacy) were evaluated in total. The relative pKa were estimatedcompared to base LNP formulation (no added SORT lipid) when 50% ofnormalized signal was produced. The TNS assay has been historically usedto measure LNPs with a single ionizable cationic lipid and neutral(non-ionizable) helper lipids, producing a value that captures theionization behavior of a cationic lipid within a self-assembled LNP. Amodified method was used here because SORT LNPs with high percentages(>40%) of the permanently cationic lipid (e.g. DOTAP) do not buffercharge well even though they contain ionizable cationic lipid. Due tothe complexity of SORT LNPs that contain a variety of charged lipids(not a single ionizable cationic lipid as in traditional LNPs), thefocus was on the relative signal at 50%, which correlated with in vivotissue specific activity.

FIGS. 30A & 30B show inclusion of an ionizable lipid (e.g. 5A2-SC8) wasrequired for efficacy. LNPs that contained SORT lipids, but no ionizablecationic lipid were in active. (30A) Schematic illustration of SORT C2LNPs. (30B) Details of C2 and SORT lipids C2 LNPs. Ex vivo luciferaseimages showed that both DODAP and DOTAP failed to enable significantmRNA delivery of C2 LNPs. These results indicate that the ionizableamino lipid is required for successful mRNA delivery (0.1 mg/kg, 6 h,n=2).

FIGS. 31A-31E show SORT LNPs enabled tissue specific gene editing inTd-Tomato mice by Cre mRNA delivery. (31A) Schematic illustration showsthat delivery of Cre mRNA activates Td-Tom expression in Td-Tomtransgenic mice. (31B) mDLNP and 20% DODAP LNPs induced Td-Tomfluorescence specifically in the liver and 50% DOTAP LNPs selectivelyedited the lung. Td-Tom fluorescence of main organs was detected 2 daysfollowing IV injection of Cre mRNA-loaded LNPs (0.3 mg/kg). (31C) 30%18PA SORT LNPs induced gene editing in the spleen (note high liverbackground fluorescence in PBS injected mice). (31D) Confocal microscopywas employed to further verify effective tissue editing. Scale bars=20μm and 100 μm. (31E) FACS was used to quantify the percentage of TdTom⁺cells within defined cell type populations of the liver, lung, andspleen (day 2, 0.3 mg/kg).

FIG. 32 shows B6.Cg-Gt(ROSA)26Sor^(tm9(CAG-tdTomato)Hze)/J (Ai9) miceexhibit some autofluorescence in the absorption region for TdTom.Moreover, there is a large difference for TdTom autofluorescence betweendifferent organs (n=2). Liver and kidney show the highest signal andspleen shows the lowest. Although this does not interfere with detectionof editing in most organs (when the excitation settings are properlyadjusted to eliminate background), it does complicate detection ofspleen TdTom expression because background spleen is so much lower thanother organs.

FIGS. 33A-33C show CRISPR/Cas gene editing in the spleen was achieved inboth Td-Tom transgenic mice and wild type C57/BL6 mice by co-deliveringCas9 mRNA and sgRNA. (33A) Schematic illustration shows that co-deliveryof Cas9 mRNA and sgTom1 activates Td-Tom expression in Td-Tom mice.(33B) Td-Tom expression was induced in the spleen and liver by thespleen-targeted formulation 30% 18PA SORT LNP. Quantification datashowed that editing in the spleen was higher than in the liver. Td-Tomfluorescence of main organs was detected at day 2 after IV treatmentwith co-delivery of Cas9 mRNA and modified sgTom1 (2/1, wt/wt) at thetotal doses of 4 mg/kg. (33C) T7E1 assay indicated that specific PTENediting of spleen was obtained by co-delivery of Cas9 mRNA (IVT) andsgPTEN. C57/BL6 mice were IV injected with 30% 18PA SORT LNPs at totaldose of 4 mg/kg (Cas9 mRNA/sgPTEN, 2/1, wt/wt), and PTEN editing wasdetected at day 2. In this case, no liver editing was observed,suggesting that spleen-specific editing can be achieved.

FIG. 34 shows DODAP-20 SORT LNPs achieved nearly 100% TdTom editing inhepatocytes administration of a single 0.3 mg/kg Cre mRNA dose. As shownin the flow cytometry histogram, there is full separation between TdTom−control mice and TdTom+ 20% DODAP treated mice. After liver perfusion,the resected livers of mice treated with 20% DODAP SORT LNPs weresurprisingly bright red compared to control livers. Even withoutfluorescence excitation, the livers glowed red due to completeactivation of TdTom expression. TdTom mice were injected with 0.3 mg/kgCre mRNA, then sacrificed after two days (n=3). Hepatocytes wereisolated by two-step collagenase perfusion and TdTom fluorescence wasanalyzed by flow cytometry.

FIG. 35 shows the FACS gating strategy for analysis of TdTom+ expressionin lung cells is described. Ghost Red 780 was used to distinguish liveand dead cells. EpCam+ was used to define epithelial cells, CD45+ andCD31− were used to define immune cells, and CD45− and CD31+ were used todefine endothelial cells. Gates for Td-Tom+ in cell types were drawnbased on PBS injected control mice. Td-Tom mice were injected with CremRNA formulations and Td-Tom+ in given cell types was detected by flowafter two days (n=3).

FIG. 36 shows the FACS gating strategy for analysis of TdTom+ expressionin splenic cells is described. Ghost Red 780 was used to distinguishlive and dead cells. CD44+ was used to distinguish immune cells, thenCD3+ and CD11b− were used for T cells, CD3− and CD11b+ were used formacrophage cells, CD19+ and CD11b− were used for B cells. Gates forTd-Tom+ in cell types were drawn based on PBS injected control mice.Td-Tom mice were injected with Cre mRNA formulations and Td-Tomato+ ingiven cell types was detected by flow after two days (n=3).

FIG. 37A-37G show SORT LNPs mediated tissue-specific CRISPR/Cas geneediting of Td-Tom transgenic mice and C57/BL6 wild type mice byco-delivering Cas9 mRNA and sgRNA and by delivering Cas9 RNPs. (37A)Schematic illustration shows that co-delivery of Cas9 mRNA (or Cas9protein) and sgTom1 activates Td-Tom expression in Td-Tom transgenicmice. (37B) mDLNP and 20% DODAP LNPs induced Td-Tom fluorescencespecifically in the liver and 50% DOTAP LNPs selectively edited thelung. Td-Tom fluorescence was detected 10 days following IV injection ofCas9 mRNA and modified sgTom1 (4/1, wt/wt) at a total dose of 2.5 mg/kg.(37C) tdTom expression was confirmed by confocal imaging of tissuesections. Scale bars=20 μm and 100 μm (37D) Cas9 mRNA and sgPTEN wereco-delivered in SORT LNPs to selectively edit the liver, lung, andspleen of C57/BL6 mice (total dose of 2.5 mg/kg (Cas9 mRNA/sgPTEN, 4/1,wt/wt; measured 10 days following a single injection). The T7E1 assayindicated that tissue specific PTEN editing was achieved. (37E) H&Esections and IHC further confirmed successful PTEN editing. Clearcytoplasm indicated lipid accumulation in H&E sections and PTEN loss inIHC images. Scale bar=60 μm. (37F) Delivery of Cas9/sgTom1ribonucleoprotein (RNP) complexes in 7% DOTAP or 55% DOTAP SORT LNPsinduced Td-Tom fluorescence specifically in the liver and lungs,respectively. Td-Tom fluorescence was detected 7 days following IVinjection of Cas9/sgTom1 RNPs at a dose of 1.5 mg/kg sgTom1. tdTomexpression was confirmed by confocal imaging of tissue sections. Scalebars=20 μm and 100 μm (37G) Liver- and lung-tropic SORT LNPs alsodelivered Cas9/sgPTEN RNPs to selectively edit the liver and lungsC57/BL6 mice (1.5 mg/kg sgPTEN; measured 7 days following a singleinjection). The T7E1 assay indicated that tissue specific PTEN editingwas achieved.

FIG. 38 shows IVT Cas9 mRNA was evaluated by western blot. 293T cellswere seeded in 12-well plate the day before transfection, cells weretreated for 24 h by each condition before performing western blot. Cas9pDNA was delivered by Lipofectamine 2000 and mRNAs were delivered bymDLNPs.

FIGS. 39A & 39B show weight ratios of IVT Cas9 mRNA to sgTom1 wereoptimized via Cas9 mRNA and sgRNA co-delivery strategy. (39A) Schematicillustration shows that co-delivery of Cas9 mRNA and sgTom1 activatesTd-Tom expression in transgenic mouse. (39B) Td-Tom fluorescence ofmajor organs was imaged at day 7 after IV injection, indicating that 2/1of Cas9/sgTom1 (wt/wt) was optimal. The total RNA dose was 1 mg/kg, IVTCas9 mRNA and modified sgTom1 were co-encapsulated by mDLNPs.

FIGS. 40A-40I show a modular approach was developed to enable systemicnanoparticle delivery of CRISPR/Cas9 ribonucleoproteins (RNPs) fortissue-specific genome editing. (40A) Addition of a permanently cationicsupplemental component (e.g. DOTAP) into traditional LNP formulationsenabled encapsulation and protection of Cas9/sgRNA complexes usingneutral buffers during nanoparticle formation. Precise tuning of theDOTAP percentage mediated tissue-specific gene editing. (40B) Sizedistribution of Cas9/sgLuc RNPs prepared in PBS buffer (pH 7.4) andcitrate buffer (pH 4.0). The size increase is likely due todenaturization. (40C) Size distribution of 5A2-DOT-10 encapsulatingCas9/sgLuc RNPs prepared in PBS and citrate buffer. 5A2-DOT-10 preparedwithout RNPs was used as control. (40D) Size distribution of Cas9/sgRNARNPs with Cas9/sgLuc molar ratio of 1/1, 1/3 and 1/5. (40E) Sizedistribution of 5A2-DOT-10 encapsulating Cas9/sgLuc with molar ratio of1/1, 1/3 and 1/5. (40F) Zeta potential of Cas9/sgRNA RNPs showingdecreasing charge. (40G) No significant difference of zeta potential wasobserved for 5A2-DOT-10 encapsulating Cas9/sgLuc with different molarratios. (40H) Time-dependent cellular uptake of 5A2-DOT-10 LNPsencapsulating EGFP-fused Cas9/sgRNAs showing cytoplasmic release andgradual entry into the nucleus. (40I) Inhibition of 5A2-DOT-10 LNPuptake was studied using specific endocytosis inhibitors. AMI: inhibitorof macropinocytosis; CMZ: inhibitor of clathrin-mediated endocytosis;GEN: inhibitor of caveolae-mediated endocytosis; MPCD: lipidrafts-mediated endocytosis; 4 degree: energy mediated endocytosis.

FIGS. 41A-C show (41A) table of 5A2-DOT-X LNPs showing the molar ratiosand percentages used to formulate 5A2-DOT-5 (5 mole % DOTAP),5A2-DOT-10, 5A2-DOT-20, 5A2-DOT-30, 5A2-DOT-40, 5A2-DOT-50, and5A2-DOT-60 (60 mole % DOTAP) LNPs. A total lipids/sgRNA ratio of 40:1(wt.) was used for all LNPs. (41B) Gene editing in HeLa-Luc cellsfollowing treatment with different 5A2-DOT-X Cas9/sgLuc RNP formulationswas detected using the T7EI assay. (41C) Gene editing was analyzed usingSanger sequencing and ICE analysis.

FIG. 42 show representative TEM images of 5A2-DOT-10 encapsulatingCas9/sgLuc RNP complexes with molar ratio of 1/3. 5A2-DOT-10 Cas9/sgLucwas prepared at total lipid concentration of 2 mg/mL in PBS buffer. 3 μLof the nanoparticle solutions was dropped onto carbon TEM grids andallowed to deposit for 1 min before blotting with filter paper. Then theTEM grids were imaged using Transmission Electron Microscopy (FEI TecnaiG2 Spirit Biotwin).

FIG. 43 shows confocal images showing cellular uptake of PBS (control),free Cas9/sgLuc complexes (control), and 5A2-DOT-10 Cas9/sgLuc inHela-Luc cells 20 hr following treatment. Cas9-EGFP fusion protein wasused to track the subcellular distribution of Cas9/sgRNA complexes. TheCas9/sgLuc complexes exhibited no detectable green fluorescence abovebackground (PBS) inside cells, while bright green signals were detectedafter treated with 5A2-DOT-10.

FIGS. 44A-44H show gene editing occurs quickly and effectively in vitro.(44A) T7EI cleavage assay of DNA isolated from HeLa-Luc cells treatedwith various nanoparticles and controls. Highly effective gene editingwas mediated by 5A2-DOT-10 delivering Cas9/sgLuc RNPs (1/3 and 1/5).Indels (%) at Luc loci was quantified by ICE analysis. Note that geneediting was 0% for LNPs prepared using low pH citrate buffer (thecurrently used and established method). (44B) Fluorescence microscopyimages of HeLa-GFP cells after treatment with various formulations.Scale bar=100 μm. 5A2-DOT-10 Cas9/sgGFP treatment significantlydecreased GFP fluorescence. (44C) Flow cytometry analysis of HeLa-GFPcells after treatment with various formulations. The peak of GFPpositive cells shifted completely to the left only for the 5A2-DOT-10Cas9/sgGFP group, indicating almost all GFP positive cells went dark.(44D) Time-dependent GFP fluorescence intensity of HeLa-GFP cells aftervarious treatments. Permanent GFP fluorescence loss after day 2 wasobserved with 5A2-DOT-10 Cas9/sgGFP treatment, while ICE analysis ofSanger sequencing data showed that indels was maintained at higher than90% after day 2. (44E & 44F) Mean Fluorescence Intensity (%) of HeLa-GFPcells after treatment with Cas9/sgGFP alone, Cas9/sgGFP-loaded 5A2-SC8,C12-200, DLin-MC3-DMA LNP formulations containing 10% supplementalDOTAP, Cas9/sgGFP-loaded traditional C12-200 and DLin-MC3-DMA LNPsnanoformulations, and Cas9/sgGFP-loaded RNAiMAX. The GFP fluorescencesignificantly decreased after treated with all three DOTAP-modifiedformulations. The ICE analysis of Sanger sequencing data furtherconfirmed the highest gene editing efficiency was with 5A2-DOT-10 LNPs.Mean±s.e.m. (n=3). Statistical significance was determined using atwo-sided Student's t-test. †: t value=42.69, degrees of freedom (df)=4(P<0.0001); ††: t value=16.75, degrees of freedom (df)=4 (P<0.0001);†††: t value=37.53, degrees of freedom (df)=4 (P<0.0001). P value<0.05was considered statistically significant. (44G) 5A2-DOT-10 Cas9/sgGFPLNPs were stored at 4° C. for 2 months. The nanoparticle diameter andPDI was monitored over time. (44H) Periodic treatment of HeLa-GFP cellswith stored LNPs showed that no activity was lost, indicating long-termLNP and RNP stability and potential for translation. All cells in theexperiments above were treated with 24 nM sgRNA.

FIGS. 45A & 45B show gene editing of different nanoformulations inHela-GFP cells. (45A) Mean fluorescence intensity (%) of Hela-GFP cellsafter treated with Cas9/sgGFP alone, 5A2-DOT-10 Cas9/sgLuc, and5A2-DOT-10 Cas9/sgGFP (at total lipids/sgGFP weight ratio of 40:1).(45B) Mean fluorescence intensity (%) of Hela-GFP cells after treatmentof 5A2-DOT-10 Cas9/sgGFP prepared with total lipids/sgGFP weight ratioat 10:1, 20:1, 30:1 and 40:1.

FIGS. 46A-46K show the generalizable RNP delivery strategy (FIG. 40A) isuniversal for ionizable cationic lipid nanoparticles (DLNPs, LLNPs,SNALPs) and for other cationic lipids that are positively charged at pH7.4 and for other neutral buffers. (46A) Scheme of LNP formulation withdifferent ionizable lipids. (46B) Details of LNP formulations withdifferent ionizable lipids, including determinate molar ratio andpercentage of each component, and the weight ratio of total lipids tosgRNA. (46C) Chemical structures of ionizable cationic lipids used informulations, including 5A2-SC8, C12-200, and Dlin-MC3-DMA. (46D) MeanFluorescence Intensity (%) of HeLa-GFP cells following treatment withCas9/sgGFP RNPs encapsulated in 5A2-DOT-10, C12-200-DOT-10, andMC3-DOT-10. The GFP fluorescence significantly decreased after treatmentwith all three formulations. (46E) Scheme of LNP formulation preparationwith different permanently cationic lipids. (46F) Details of LNPformulations with different permanently cationic lipids, includingdeterminate molar ratio and percentage of each component, and the weightratio of total lipids to sgRNA. (46G) Chemical structures of permanentlycationic lipids used in formulations, including DOTAP, DDAB, and EPC.(46H) Mean Fluorescence Intensity (%) of HeLa-GFP cells after treatmentwith Cas9/sgGFP RNPs encapsulated in 5A2-DOT-10, 5A2-DDAB-10, and5A2-EPC-10. Instead of DOTAP, other cationic lipids (DDAB and EPC) werealso able to achieve efficient gene editing. (46I) Scheme of LNPformulation in different buffers. (46J) Mean Fluorescence Intensity (%)of HeLa-GFP cells after treatment with 5A2-DOT-10 formulated usingdifferent buffers, including PBS, Opti-MEM, HEPES, and Citrate Buffer.Neutral buffer was required for RNP encapsulation and delivery. (46K)Indels (%) at GFP loci in genomic DNA isolated from HeLa-GFP cells aftertreatment with 5A2-DOT-10 Cas9/sgGFP LNPs prepared using differentbuffers were measured using ICE analysis. All neutral buffers showedhigh gene editing in cells, demonstrating the importance of neutralbuffers in nanoparticle preparation. Please note that FIGS. 44E and 44Fhave been reproduced above in FIG. 45 to assemble relevant data togetherfor enhanced clarity.

FIGS. 47A-47J show highly efficient multiplexed genome editing wasachieved in vivo. (47A) Schematic illustration shows how delivery ofCas9/sgTOM RNPs activates Td-Tom expression in Td-Tomato transgenicmice. 5A2-DOT-X LNPs were injected into Td-Tom mice locally (viaintra-muscle or intra-brain injections) and systemically (via i.v.injection through tail vein). In vivo imaging of Td-Tom mice afterintra-muscle (1 mg/kg sgTom) (47B) or intra-brain (0.15 mg/kg sgTOM)(47D) injection of 5A2-DOT-10 Cas9/sgTOM showed bright red fluorescencein the leg muscle or brain tissue (respectively). Successful CRISPR/Casgene editing was further confirmed by confocal imaging of (47C) muscleand (47E) brain tissue sections. 5A2-DOT-10 enabled higher gene editingefficiency than positive control RNAiMAX, which has previously been usedfor local RNP injections. (47F) In vivo imaging of Td-Tom mice afterintravenous (IV) injection of 5A2-DOT-X Cas9/sgTOM LNPs with differentmolar percentages of DOTAP. Td-Tom fluorescence, as a downstream readoutof DNA editing, showed that low DOTAP percentages facilitated liverediting while high DOTAP percentages facilitated lung editing (1.5 mg/kgsgTOM, IV). (47G) Successful CRISPR/Cas gene editing was furtherconfirmed by confocal imaging. (47H) The T7EI cleavage assay wasperformed on DNA isolated from liver and lung tissues after systemic IVtreatment with 5A2-DOT-5, 5A2-DOT-10, 5A2-DOT-50, and 5A2-DOT-60encapsulating Cas9/sgPTEN. Indels (%) was calculated and reported. (47I)5A2-DOT-50 LNPs containing pooled sgRNAs for 6 targets (sgTOM, sgP53,sgPTEN, sgEml4, sgALK, and sgRB1) (5A2-DOT-50-Pool) were administered totd-Tom mice IV at total RNA dose of 2 mg/kg (0.33 mg/kg each sgRNA).Gene editing at the TOM loci was confirmed by in vivo imaging and (47J)editing of the other 5 loci was confirmed using the T7EI cleavage assayon lung tissues.

FIGS. 48A-48H show 5A2-DOT-X LNPs simplify generation of complex mousemodels. (48A) To create an in situ liver-specific cancer model,5A2-DOT-5 LNPs encapsulating Cas9/sgP53/sgPTEN/sgRB1 RNPs were injectedinto adult C57BL/6 mice weekly (3 injections, 2.5 mg/kg total sgRNA, IV,n=4). After 12, 15, and 20 weeks, mice were sacrificed and livers werecollected to analyze tumor generation. (48B) T7EI cleavage results fromgenomic DNA extracted from livers confirmed gene editing occurred at allthree loci. (48C) Representative photograph of a mouse liver containingtumors excised 20 weeks after injection. (48D) H&E and Ki67 stainingfurther confirmed progressive tumor formation. Higher tumorproliferation biomarker Ki67 expression was detected in tumor lesions.Scale bar=100 μm. (48E) To create an in situ lung-specific cancer model,5A2-DOT-50 LNPs encapsulating Cas9/sgEml4/sgAlk RNPs were injected intoadult C57BL/6 mice once (2 mg/kg) or twice (1.5 mg/kg weekly for 2weeks) (IV, n=5). After 10, 16, and 24 weeks, mice were sacrificed andlungs were collected to analyze tumor generation. (48F) T7EI cleavageresults from genomic DNA extracted from lungs confirmed gene editingoccurred at loci of Eml4 and Alk. PCR amplicons of Eml4-Alkrearrangements were also detected in all lungs treated with 5A2-DOT-50LNPs. (48G) Eml4-Alk rearrangements were further confirmed bysub-cloning and DNA sequencing (Predicted=SEQ ID NO: 50; Clone1=SEQ IDNO: 51; Clone2=SEQ ID NO: 52; Clone3=SEQ ID NO: 53; Clone4=SEQ ID NO:54; Clone5=SEQ ID NO: 55; Clone6=SEQ ID NO: 56; Clone7=SEQ ID NO: 57;Clone8=SEQ ID NO: 58). (48H) H&E and Ki67 staining further confirmedprogressive tumor formation. Higher tumor proliferation biomarker Ki67expression was detected in lung tumor lesions. Scale bar=100 μm.

FIGS. 49A & 49B show gene editing efficiency of unmodified sgRNAsynthesized by in vitro transcription (IVT) comparing to chemicallymodified and synthesized sgRNA (2′-methyl 3′-phosphorothioatemodifications in the first and last 3 nucleotides). (49A) Relativeluciferase activity in Hela-Luc-Cas9 cells after treatment with IVTsgRNA and chemically modified sgRNA encapsulated inside nanoparticles.(49B) T7EI assay detecting gene editing efficiency of Cas9/IVT sgRNA andCas9/chemically modified sgRNA encapsulated nanoparticles. Cleavagebands at 536 bp and 184 bp were observed clearly with modified sgRNAtreatment group.

FIG. 50 shows gene editing of P53, PTEN and RB1 genes in mouse liverafter treatment with 5A2-DOT-5 LNPs encapsulatingCas9/sgP53/sgPTEN/sgRB1 RNPs. T7EI assay detecting the gene editing ofliver genomic DNA at PTEN, P53 and RB1 genome loci, after treatmentweekly for two weeks. PBS treatment group was used as control. Cleavagebands were detected at 261 bp and 215 bp of PCR amplicons targeting P53;cleavage bands were detected at 345 bp and 293 bp of PCR ampliconstargeting PTEN; cleavage bands were detected at 395 bp and 207 bp of PCRamplicons targeting RB1.

FIG. 51 shows T7EI assay detecting the gene editing of P53, PTEN and RB1genes in mouse liver after treatment with 5A2-DOT-5 LNPs encapsulatingCas9/sgP53/sgPTEN/sgRB1 RNPs. PBS treatment and 5A2-DOT-5 only (noCas9/sgRNA) treatment groups were used as control. The T7EI result ofgenome DNA extracted from tumor of mice after treated with 5A2-DOT-5LNPs encapsulating Cas9/sgP53/sgPTEN/sgRB1 RNPs for 20 weeksdemonstrated the tumor generation was induced by knockout of these threegenes, as cleavage bands were detected at all three genome loci.

FIG. 52 shows representative photograph of a mouse liver and excisedtumors excised from a mouse in the group treated with 5A2-DOT-5 LNPsencapsulating Cas9/sgP53/sgPTEN/sgRB1 RNPs for 15 weeks.

FIGS. 53A-53C show H&E and Ki67 staining images of mouse liversfollowing treatment with 5A2-DOT-5 LNPs only (no Cas9/sgRNA) (control)for 15 weeks and 20 weeks (53A), tumors excised from a mouse in thegroup treated with 5A2-DOT-5 LNPs encapsulating Cas9/sgP53/sgPTEN/sgRB1RNPs for 20 weeks (53B). No morphological changes were detected with5A2-DOT-5 LNPs only treatments, suggesting nanovectors alone could notlead to tumors. Scale bar: 100 μm. (53C) Large view images of mouseliver tumor generation after treated with 5A2-DOT-5 LNPs encapsulatingCas9/sgP53/sgPTEN/sgRB1 RNPs for 20 weeks. Scale bar: 500 μm.

FIGS. 54A-54D show generation of Eml4-Alk rearrangements in mouse lungsafter treatment with 5A2-DOT-50 LNPs encapsulating Cas9/sgEml4/sgAlkRNPs for 7 days (2 mg/kg of total sgRNA). Eml4 editing (54A) and Alkediting (54B) were detected in genomic DNA extracted from mouse lungs byT7EI assay. (54C) PCR analysis was performed on genomic DNA extractedfrom mouse lungs to determine Eml4-Alk inversion. (54D) The PCRamplicons were sub-cloned and the sequences of 6 independent clones werelisted, together with a representative chromatogram presented on theupper panel. The chromatogram was exactly the same as predicted forEml4-Alk rearrangement. (Predicted=SEQ ID NO: 59; Clone1=SEQ ID NO: 59;Clone2=SEQ ID NO: 60; Clone4=SEQ ID NO: 61; Clone5=SEQ ID NO: 61;Clone6=SEQ ID NO: 62)

FIG. 55 shows H&E and Ki67 staining images of mouse livers treated with5A2-DOT-50 LNPs Only (no Cas9/sgRNA) for 10 weeks and 16 weeks (LNP doseequal to 1 mg/kg of total sgRNA). No morphological changes were detectedin 5A2-DOT-50 LNPs Only injected animals. Scale bar: 100 μm.

FIG. 56 shows large view images of mouse lung tumor generation aftertreated with 5A2-DOT-50 LNPs encapsulating Cas9/sgEml4/sgAlk RNPs for 24weeks. Scale bar: 500 μm. Several tumor lesions (highlighted) wereobserved from both H&E and Ki67 staining images.

FIG. 57 shows 5A2-DOT-10 LNPs could deliver ovalbumin (OVA) proteinefficiently into the cytoplasm of HeLa-Luc cells. Cells were treatedwith free rhodamine-labeled OVA protein and 5A2-DOT-10 LNPsencapsulating rhodamine-labeled OVA for 22 h before imaging by confocalmicroscopy.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Described herein is a lipid nanoparticle (LNP), composed of 1) apermanently cationic lipid, 2) an ionizable cationic lipid, and 3) aphospholipid and may optionally contain either cholesterol and lipid PEGor both. Inclusion of the permantly cationic lipid serves to direct theLNP to a specific organ such as the lungs, the lymph nodes, or thespleen. The data presented herein indicate that this effect is general,and the components are modular with each category indicating that5A2-SC8 can be replaced by any ionizable cationic lipid, DOTAP can bereplaced by any cationic lipid, and DOPE can be replaced by anyphospholipid. In some embodiments, cholesterol and lipid PEG are alsoincluded, but formulations without cholesterol or lipid PEG arefeasible. These carriers can deliver mRNA, sgRNA, and proteins tospecific organs in vivo, thus solving a major challenge.

A. CHEMICAL DEFINITIONS

When used in the context of a chemical group: “hydrogen” means —H;“hydroxy” means —OH; “oxo” means ═O; “carbonyl” means —C(═O)—; “carboxy”means —C(═O)OH (also written as —COOH or —CO₂H); “halo” meansindependently —F, —Cl, —Br or —I; “amino” means —NH₂; “hydroxyamino”means —NHOH; “nitro” means —NO₂; imino means=NH; “cyano” means —CN;“isocyanate” means —N═C═O; “azido” means —N₃; in a monovalent context“phosphate” means —OP(O)(OH)₂ or a deprotonated form thereof; in adivalent context “phosphate” means —OP(O)(OH)O— or a deprotonated formthereof, “mercapto” means —SH; and “thio” means ═S; “sulfonyl” means—S(O)₂—; “hydroxysulfonyl” means —S(O)₂OH; “sulfonamide” means—S(O)₂NH₂; and “sulfinyl” means —S(O)—.

In the context of chemical formulas, the symbol “—” means a single bond,“═” means a double bond, and “≡” means triple bond. The symbol “

” represents an optional bond, which if present is either single ordouble. The symbol “

” represents a single bond or a double bond. Thus, for example, theformula

includes

And it is understood that no one such ring atom forms part of more thanone double bond. Furthermore, it is noted that the covalent bond symbol“—”, when connecting one or two stereogenic atoms, does not indicate anypreferred stereochemistry. Instead, it covers all stereoisomers as wellas mixtures thereof. The symbol “

”, when drawn perpendicularly across a bond (e.g.

for methyl) indicates a point of attachment of the group. It is notedthat the point of attachment is typically only identified in this mannerfor larger groups in order to assist the reader in unambiguouslyidentifying a point of attachment. The symbol “

” means a single bond where the group attached to the thick end of thewedge is “out of the page.” The symbol “

” means a single bond where the group attached to the thick end of thewedge is “into the page”. The symbol “

” means a single bond where the geometry around a double bond (e.g.,either E or Z) is undefined. Both options, as well as combinationsthereof are therefore intended. Any undefined valency on an atom of astructure shown in this application implicitly represents a hydrogenatom bonded to that atom. A bold dot on a carbon atom indicates that thehydrogen attached to that carbon is oriented out of the plane of thepaper.

When a group “R” is depicted as a “floating group” on a ring system, forexample, in the formula:

then R may replace any hydrogen atom attached to any of the ring atoms,including a depicted, implied, or expressly defined hydrogen, so long asa stable structure is formed. When a group “R” is depicted as a“floating group” on a fused ring system, as for example in the formula:

then R may replace any hydrogen attached to any of the ring atoms ofeither of the fused rings unless specified otherwise. Replaceablehydrogens include depicted hydrogens (e.g., the hydrogen attached to thenitrogen in the formula above), implied hydrogens (e.g., a hydrogen ofthe formula above that is not shown but understood to be present),expressly defined hydrogens, and optional hydrogens whose presencedepends on the identity of a ring atom (e.g., a hydrogen attached togroup X, when X equals —CH—), so long as a stable structure is formed.In the example depicted, R may reside on either the 5-membered or the6-membered ring of the fused ring system. In the formula above, thesubscript letter “y” immediately following the group “R” enclosed inparentheses, represents a numeric variable. Unless specified otherwise,this variable can be 0, 1, 2, or any integer greater than 2, onlylimited by the maximum number of replaceable hydrogen atoms of the ringor ring system.

For the chemical groups and compound classes, the number of carbon atomsin the group or class is as indicated as follows: “Cn” defines the exactnumber (n) of carbon atoms in the group/class. “C≤n” defines the maximumnumber (n) of carbon atoms that can be in the group/class, with theminimum number as small as possible for the group/class in question,e.g., it is understood that the minimum number of carbon atoms in thegroup “alkenyl_((C≤8))” or the class “alkene_((C≤8))” is two. Comparewith “alkoxy_((C≤1))”, which designates alkoxy groups having from 1 to10 carbon atoms. “Cn-n” defines both the minimum (n) and maximum number(n′) of carbon atoms in the group. Thus, “alkyl_((C2-10))” designatesthose alkyl groups having from 2 to 10 carbon atoms. These carbon numberindicators may precede or follow the chemical groups or class itmodifies and it may or may not be enclosed in parenthesis, withoutsignifying any change in meaning. Thus, the terms “C5 olefin”,“C5-olefin”, “olefin_((C5))”, and “olefin_(C5)” are all synonymous.

The term “saturated” when used to modify a compound or chemical groupmeans the compound or chemical group has no carbon-carbon double and nocarbon-carbon triple bonds, except as noted below. When the term is usedto modify an atom, it means that the atom is not part of any double ortriple bond. In the case of substituted versions of saturated groups,one or more carbon oxygen double bond or a carbon nitrogen double bondmay be present. And when such a bond is present, then carbon-carbondouble bonds that may occur as part of keto-enol tautomerism orimine/enamine tautomerism are not precluded. When the term “saturated”is used to modify a solution of a substance, it means that no more ofthat substance can dissolve in that solution.

The term “aliphatic” when used without the “substituted” modifiersignifies that the compound or chemical group so modified is an acyclicor cyclic, but non-aromatic hydrocarbon compound or group. In aliphaticcompounds/groups, the carbon atoms can be joined together in straightchains, branched chains, or non-aromatic rings (alicyclic). Aliphaticcompounds/groups can be saturated, that is joined by singlecarbon-carbon bonds (alkanes/alkyl), or unsaturated, with one or morecarbon-carbon double bonds (alkenes/alkenyl) or with one or morecarbon-carbon triple bonds (alkynes/alkynyl).

The term “aromatic” when used to modify a compound or a chemical groupatom means the compound or chemical group contains a planar unsaturatedring of atoms that is stabilized by an interaction of the bonds formingthe ring.

The term “alkyl” when used without the “substituted” modifier refers toa monovalent saturated aliphatic group with a carbon atom as the pointof attachment, a linear or branched acyclic structure, and no atomsother than carbon and hydrogen. The groups —CH₃ (Me), —CH₂CH₃ (Et),—CH₂CH₂CH₃ (n-Pr or propyl), —CH(CH₃)₂(i-Pr, ¹Pr or isopropyl),—CH₂CH₂CH₂CH₃ (n-Bu), —CH(CH₃)CH₂CH₃ (sec-butyl), —CH₂CH(CH₃)₂(isobutyl), —C(CH₃)₃ (tert-butyl, t-butyl, t-Bu or ^(r)Bu), and—CH₂C(CH₃)₃ (neo-pentyl) are non-limiting examples of alkyl groups. Theterm “alkanediyl” when used without the “substituted” modifier refers toa divalent saturated aliphatic group, with one or two saturated carbonatom(s) as the point(s) of attachment, a linear or branched acyclicstructure, no carbon-carbon double or triple bonds, and no atoms otherthan carbon and hydrogen. The groups —CH₂— (methylene), —CH₂CH₂—,—CH₂C(CH₃)₂CH₂—, and —CH₂CH₂CH₂— are non-limiting examples of alkanediylgroups. An “alkane” refers to the class of compounds having the formulaH—R, wherein R is alkyl as this term is defined above. When any of theseterms is used with the “substituted” modifier one or more hydrogen atomhas been independently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂,—CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂. The following groups are non-limiting examplesof substituted alkyl groups: —CH₂OH, —CH₂Cl, —CF₃, —CH₂CN, —CH₂C(O)OH,—CH₂C(O)OCH₃, —CH₂C(O)NH₂, —CH₂C(O)CH₃, —CH₂OCH₃, —CH₂OC(O)CH₃, —CH₂NH₂,—CH₂N(CH₃)₂, and —CH₂CH₂C₁. The term “haloalkyl” is a subset ofsubstituted alkyl, in which the hydrogen atom replacement is limited tohalo (i.e. —F, —Cl, —Br, or —I) such that no other atoms aside fromcarbon, hydrogen and halogen are present. The group, —CH₂C₁ is anon-limiting example of a haloalkyl. The term “fluoroalkyl” is a subsetof substituted alkyl, in which the hydrogen atom replacement is limitedto fluoro such that no other atoms aside from carbon, hydrogen andfluorine are present. The groups —CH₂F, —CF₃, and —CH₂CF₃ arenon-limiting examples of fluoroalkyl groups.

The term “cycloalkyl” when used without the “substituted” modifierrefers to a monovalent saturated aliphatic group with a carbon atom asthe point of attachment, said carbon atom forming part of one or morenon-aromatic ring structures, no carbon-carbon double or triple bonds,and no atoms other than carbon and hydrogen. Non-limiting examplesinclude: —CH(CH₂)₂ (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl(Cy). The term “cycloalkanediyl” when used without the “substituted”modifier refers to a divalent saturated aliphatic group with two carbonatoms as points of attachment, no carbon-carbon double or triple bonds,and no atoms other than carbon and hydrogen. The group

is a non-limiting example of cycloalkanediyl group. A “cycloalkane”refers to the class of compounds having the formula H—R, wherein R iscycloalkyl as this term is defined above. When any of these terms isused with the “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂.

The term “alkenyl” when used without the “substituted” modifier refersto an monovalent unsaturated aliphatic group with a carbon atom as thepoint of attachment, a linear or branched, acyclic structure, at leastone nonaromatic carbon-carbon double bond, no carbon-carbon triplebonds, and no atoms other than carbon and hydrogen. Non-limitingexamples include: —CH═CH₂ (vinyl), —CH═CHCH₃, —CH═CHCH₂CH₃, —CH₂CH═CH₂(allyl), —CH₂CH═CHCH₃, and —CH═CHCH═CH₂. The term “alkenediyl” when usedwithout the “substituted” modifier refers to a divalent unsaturatedaliphatic group, with two carbon atoms as points of attachment, a linearor branched, a linear or branched acyclic structure, at least onenonaromatic carbon-carbon double bond, no carbon-carbon triple bonds,and no atoms other than carbon and hydrogen. The groups —CH═CH—,—CH═C(CH₃)CH₂—, —CH═CHCH₂—, and —CH₂CH═CHCH₂— are non-limiting examplesof alkenediyl groups. It is noted that while the alkenediyl group isaliphatic, once connected at both ends, this group is not precluded fromforming part of an aromatic structure. The terms “alkene” and “olefin”are synonymous and refer to the class of compounds having the formulaH—R, wherein R is alkenyl as this term is defined above. Similarly theterms “terminal alkene” and “α-olefin” are synonymous and refer to analkene having just one carbon-carbon double bond, wherein that bond ispart of a vinyl group at an end of the molecule. When any of these termsare used with the “substituted” modifier one or more hydrogen atom hasbeen independently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂. The groups —CH═CHF, —CH═CHCl and —CH═CHBr arenon-limiting examples of substituted alkenyl groups.

The term “alkynyl” when used without the “substituted” modifier refersto a monovalent unsaturated aliphatic group with a carbon atom as thepoint of attachment, a linear or branched acyclic structure, at leastone carbon-carbon triple bond, and no atoms other than carbon andhydrogen. As used herein, the term alkynyl does not preclude thepresence of one or more non-aromatic carbon-carbon double bonds. Thegroups —C≡CH, —C≡CCH₃, and —CH₂C≡CCH₃ are non-limiting examples ofalkynyl groups. An “alkyne” refers to the class of compounds having theformula H—R, wherein R is alkynyl. When any of these terms are used withthe “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂.

The term “aryl” when used without the “substituted” modifier refers to amonovalent unsaturated aromatic group with an aromatic carbon atom asthe point of attachment, said carbon atom forming part of a one or moresix-membered aromatic ring structure, wherein the ring atoms are allcarbon, and wherein the group consists of no atoms other than carbon andhydrogen. If more than one ring is present, the rings may be fused orunfused. As used herein, the term does not preclude the presence of oneor more alkyl or aralkyl groups (carbon number limitation permitting)attached to the first aromatic ring or any additional aromatic ringpresent. Non-limiting examples of aryl groups include phenyl (Ph),methylphenyl, (dimethyl)phenyl, —C₆H₄CH₂CH₃ (ethylphenyl), naphthyl, anda monovalent group derived from biphenyl. The term “arenediyl” when usedwithout the “substituted” modifier refers to a divalent aromatic groupwith two aromatic carbon atoms as points of attachment, said carbonatoms forming part of one or more six-membered aromatic ringstructure(s) wherein the ring atoms are all carbon, and wherein themonovalent group consists of no atoms other than carbon and hydrogen. Asused herein, the term does not preclude the presence of one or morealkyl, aryl or aralkyl groups (carbon number limitation permitting)attached to the first aromatic ring or any additional aromatic ringpresent. If more than one ring is present, the rings may be fused orunfused. Unfused rings may be connected via one or more of thefollowing: a covalent bond, alkanediyl, or alkenediyl groups (carbonnumber limitation permitting). Non-limiting examples of arenediyl groupsinclude:

An “arene” refers to the class of compounds having the formula H—R,wherein R is aryl as that term is defined above. Benzene and toluene arenon-limiting examples of arenes. When any of these terms are used withthe “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂.

The term “aralkyl” when used without the “substituted” modifier refersto the monovalent group -alkanediyl-aryl, in which the terms alkanediyland aryl are each used in a manner consistent with the definitionsprovided above. Non-limiting examples are: phenylmethyl (benzyl, Bn) and2-phenyl-ethyl. When the term aralkyl is used with the “substituted”modifier one or more hydrogen atom from the alkanediyl and/or the arylgroup has been independently replaced by —OH, —F, —Cl, —Br, —I, —NH₂,—NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃,—NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃,—NHC(O)CH₃, —S(O)₂OH, or —S(O)₂NH₂. Non-limiting examples of substitutedaralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-1-yl.

The term “heteroaryl” when used without the “substituted” modifierrefers to a monovalent aromatic group with an aromatic carbon atom ornitrogen atom as the point of attachment, said carbon atom or nitrogenatom forming part of one or more aromatic ring structures wherein atleast one of the ring atoms is nitrogen, oxygen or sulfur, and whereinthe heteroaryl group consists of no atoms other than carbon, hydrogen,aromatic nitrogen, aromatic oxygen and aromatic sulfur. Heteroaryl ringsmay contain 1, 2, 3, or 4 ring atoms selected from are nitrogen, oxygen,and sulfur. If more than one ring is present, the rings may be fused orunfused. As used herein, the term does not preclude the presence of oneor more alkyl, aryl, and/or aralkyl groups (carbon number limitationpermitting) attached to the aromatic ring or aromatic ring system.Non-limiting examples of heteroaryl groups include furanyl, imidazolyl,indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl,phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl,quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl,thienyl, and triazolyl. The term “N-heteroaryl” refers to a heteroarylgroup with a nitrogen atom as the point of attachment. The term“heteroarenediyl” when used without the “substituted” modifier refers toan divalent aromatic group, with two aromatic carbon atoms, two aromaticnitrogen atoms, or one aromatic carbon atom and one aromatic nitrogenatom as the two points of attachment, said atoms forming part of one ormore aromatic ring structure(s) wherein at least one of the ring atomsis nitrogen, oxygen or sulfur, and wherein the divalent group consistsof no atoms other than carbon, hydrogen, aromatic nitrogen, aromaticoxygen and aromatic sulfur. If more than one ring is present, the ringsmay be fused or unfused. Unfused rings may be connected via one or moreof the following: a covalent bond, alkanediyl, or alkenediyl groups(carbon number limitation permitting). As used herein, the term does notpreclude the presence of one or more alkyl, aryl, and/or aralkyl groups(carbon number limitation permitting) attached to the aromatic ring oraromatic ring system. Non-limiting examples of heteroarenediyl groupsinclude:

A “heteroarene” refers to the class of compounds having the formula H—R,wherein R is heteroaryl. Pyridine and quinoline are non-limitingexamples of heteroarenes. When these terms are used with the“substituted” modifier one or more hydrogen atom has been independentlyreplaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH,—OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂,—C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃, —S(O)₂OH, or —S(O)₂NH₂.

The term “heterocycloalkyl” when used without the “substituted” modifierrefers to a monovalent non-aromatic group with a carbon atom or nitrogenatom as the point of attachment, said carbon atom or nitrogen atomforming part of one or more non-aromatic ring structures wherein atleast one of the ring atoms is nitrogen, oxygen or sulfur, and whereinthe heterocycloalkyl group consists of no atoms other than carbon,hydrogen, nitrogen, oxygen and sulfur. Heterocycloalkyl rings maycontain 1, 2, 3, or 4 ring atoms selected from nitrogen, oxygen, orsulfur. If more than one ring is present, the rings may be fused orunfused. As used herein, the term does not preclude the presence of oneor more alkyl groups (carbon number limitation permitting) attached tothe ring or ring system. Also, the term does not preclude the presenceof one or more double bonds in the ring or ring system, provided thatthe resulting group remains non-aromatic. Non-limiting examples ofheterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl,piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl,tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl,oxiranyl, and oxetanyl. The term “N-heterocycloalkyl” refers to aheterocycloalkyl group with a nitrogen atom as the point of attachment.N-pyrrolidinyl is an example of such a group. The term“heterocycloalkanediyl” when used without the “substituted” modifierrefers to an divalent cyclic group, with two carbon atoms, two nitrogenatoms, or one carbon atom and one nitrogen atom as the two points ofattachment, said atoms forming part of one or more ring structure(s)wherein at least one of the ring atoms is nitrogen, oxygen or sulfur,and wherein the divalent group consists of no atoms other than carbon,hydrogen, nitrogen, oxygen and sulfur. If more than one ring is present,the rings may be fused or unfused. Unfused rings may be connected viaone or more of the following: a covalent bond, alkanediyl, or alkenediylgroups (carbon number limitation permitting). As used herein, the termdoes not preclude the presence of one or more alkyl groups (carbonnumber limitation permitting) attached to the ring or ring system. Also,the term does not preclude the presence of one or more double bonds inthe ring or ring system, provided that the resulting group remainsnon-aromatic. Non-limiting examples of heterocycloalkanediyl groupsinclude:

When these terms are used with the “substituted” modifier one or morehydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I,—NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃,—NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃,—NHC(O)CH₃, —S(O)₂OH, or —S(O)₂NH₂.

The term “acyl” when used without the “substituted” modifier refers tothe group —C(O)R, in which R is a hydrogen, alkyl, cycloalkyl, alkenyl,aryl, aralkyl or heteroaryl, as those terms are defined above. Thegroups, —CHO, —C(O)CH₃ (acetyl, Ac), —C(O)CH₂CH₃, —C(O)CH₂CH₂CH₃,—C(O)CH(CH₃)₂, —C(O)CH(CH₂)₂, —C(O)C₆H₅, —C(O)C₆H₄CH₃, —C(O)CH₂C₆H₅,—C(O)(imidazolyl) are non-limiting examples of acyl groups. A “thioacyl”is defined in an analogous manner, except that the oxygen atom of thegroup —C(O)R has been replaced with a sulfur atom, —C(S)R. The term“aldehyde” corresponds to an alkane, as defined above, wherein at leastone of the hydrogen atoms has been replaced with a —CHO group. When anyof these terms are used with the “substituted” modifier one or morehydrogen atom (including a hydrogen atom directly attached to the carbonatom of the carbonyl or thiocarbonyl group, if any) has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂. The groups, —C(O)CH₂CF₃, —CO₂H (carboxyl),—CO₂CH₃ (methylcarboxyl), —CO₂CH₂CH₃, —C(O)NH₂ (carbamoyl), and—CON(CH₃)₂, are non-limiting examples of substituted acyl groups.

The term “alkoxy” when used without the “substituted” modifier refers tothe group —OR, in which R is an alkyl, as that term is defined above.Non-limiting examples include: —OCH₃ (methoxy), —OCH₂CH₃ (ethoxy),—OCH₂CH₂CH₃, —OCH(CH₃)₂ (isopropoxy), —OC(CH₃)₃ (tert-butoxy),—OCH(CH₂)₂, —O-cyclopentyl, and —O-cyclohexyl. The terms “cycloalkoxy”,“alkenyloxy”, “alkynyloxy”, “aryloxy”, “aralkoxy”, “heteroaryloxy”,“heterocycloalkoxy”, and “acyloxy”, when used without the “substituted”modifier, refers to groups, defined as —OR, in which R is cycloalkyl,alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl,respectively. The term “alkoxydiyl” refers to the divalent group—O-alkanediyl-, —O-alkanediyl-O—, or -alkanediyl-O-alkanediyl-. The term“alkylthio” and “acylthio” when used without the “substituted” modifierrefers to the group —SR, in which R is an alkyl and acyl, respectively.The term “alcohol” corresponds to an alkane, as defined above, whereinat least one of the hydrogen atoms has been replaced with a hydroxygroup. The term “ether” corresponds to an alkane, as defined above,wherein at least one of the hydrogen atoms has been replaced with analkoxy group. When any of these terms is used with the “substituted”modifier one or more hydrogen atom has been independently replaced by—OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃,—OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃,—C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃, —S(O)₂OH, or —S(O)₂NH₂.

The term “alkylamino” when used without the “substituted” modifierrefers to the group —NHR, in which R is an alkyl, as that term isdefined above. Non-limiting examples include: —NHCH₃ and —NHCH₂CH₃. Theterm “dialkylamino” when used without the “substituted” modifier refersto the group —NRR′, in which R and R′ can be the same or different alkylgroups, or R and R′ can be taken together to represent an alkanediyl.Non-limiting examples of dialkylamino groups include: —N(CH₃)₂ and—N(CH₃)(CH₂CH₃). The terms “cycloalkylamino”, “alkenylamino”,“alkynylamino”, “arylamino”, “aralkylamino”, “heteroarylamino”,“heterocycloalkylamino”, “alkoxyamino”, and “alkylsulfonylamino” whenused without the “substituted” modifier, refers to groups, defined as—NHR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl,heteroaryl, heterocycloalkyl, alkoxy, and alkylsulfonyl, respectively. Anon-limiting example of an arylamino group is —NHC₆H₅. The term“alkylaminodiyl” refers to the divalent group —NH-alkanediyl-,—NH-alkanediyl-NH—, or -alkanediyl-NH-alkanediyl-. The term “amido”(acylamino), when used without the “substituted” modifier, refers to thegroup —NHR, in which R is acyl, as that term is defined above. Anon-limiting example of an amido group is —NHC(O)CH₃. The term“alkylimino” when used without the “substituted” modifier refers to thedivalent group ═NR, in which R is an alkyl, as that term is definedabove. When any of these terms is used with the “substituted” modifierone or more hydrogen atom attached to a carbon atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂. The groups —NHC(O)OCH₃ and —NHC(O)NHCH₃ arenon-limiting examples of substituted amido groups.

The use of the word “a” or “an,” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

As used in this application, the term “average molecular weight” refersto the relationship between the number of moles of each polymer speciesand the molar mass of that species. In particular, each polymer moleculemay have different levels of polymerization and thus a different molarmass. The average molecular weight can be used to represent themolecular weight of a plurality of polymer molecules. Average molecularweight is typically synonymous with average molar mass. In particular,there are three major types of average molecular weight: number averagemolar mass, weight (mass) average molar mass, and Z-average molar mass.In the context of this application, unless otherwise specified, theaverage molecular weight represents either the number average molar massor weight average molar mass of the formula. In some embodiments, theaverage molecular weight is the number average molar mass. In someembodiments, the average molecular weight may be used to describe a PEGcomponent present in a lipid.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and also covers other unlisted steps.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult. “Effective amount,” “Therapeutically effective amount” or“pharmaceutically effective amount” when used in the context of treatinga patient or subject with a compound means that amount of the compoundwhich, when administered to a subject or patient for treating a disease,is sufficient to effect such treatment for the disease.

As used herein, the term “IC₅₀” refers to an inhibitory dose which is50% of the maximum response obtained. This quantitative measureindicates how much of a particular drug or other substance (inhibitor)is needed to inhibit a given biological, biochemical or chemical process(or component of a process, i.e. an enzyme, cell, cell receptor ormicroorganism) by half.

An “isomer” of a first compound is a separate compound in which eachmolecule contains the same constituent atoms as the first compound, butwhere the configuration of those atoms in three dimensions differs.

As used herein, the term “patient” or “subject” refers to a livingmammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat,mouse, rat, guinea pig, or transgenic species thereof. In certainembodiments, the patient or subject is a primate. Non-limiting examplesof human subjects are adults, juveniles, infants and fetuses.

As generally used herein “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues, organs, and/or bodily fluids of human beings andanimals without excessive toxicity, irritation, allergic response, orother problems or complications commensurate with a reasonablebenefit/risk ratio.

“Pharmaceutically acceptable salts” means salts of compounds of thepresent disclosure which are pharmaceutically acceptable, as definedabove, and which possess the desired pharmacological activity. Suchsalts include acid addition salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or with organic acids such as1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,2-naphthalenesulfonic acid, 3-phenylpropionic acid,4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid),4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid,aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids,aromatic sulfuric acids, benzenesulfonic acid, benzoic acid,camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid,cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid,glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid,laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelicacid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoicacid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substitutedalkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid,salicylic acid, stearic acid, succinic acid, tartaric acid,tertiarybutylacetic acid, trimethylacetic acid, and the like.Pharmaceutically acceptable salts also include base addition salts whichmay be formed when acidic protons present are capable of reacting withinorganic or organic bases. Acceptable inorganic bases include sodiumhydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide andcalcium hydroxide. Acceptable organic bases include ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine and thelike. It should be recognized that the particular anion or cationforming a part of any salt of this disclosure is not critical, so longas the salt, as a whole, is pharmacologically acceptable. Additionalexamples of pharmaceutically acceptable salts and their methods ofpreparation and use are presented in Handbook of Pharmaceutical Salts:Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag HelveticaChimica Acta, 2002).

The term “pharmaceutically acceptable carrier,” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting a chemical agent.

“Prevention” or “preventing” includes: (1) inhibiting the onset of adisease in a subject or patient which may be at risk and/or predisposedto the disease but does not yet experience or display any or all of thepathology or symptomatology of the disease, and/or (2) slowing the onsetof the pathology or symptomatology of a disease in a subject or patientwhich may be at risk and/or predisposed to the disease but does not yetexperience or display any or all of the pathology or symptomatology ofthe disease.

A “repeat unit” is the simplest structural entity of certain materials,for example, frameworks and/or polymers, whether organic, inorganic ormetal-organic. In the case of a polymer chain, repeat units are linkedtogether successively along the chain, like the beads of a necklace. Forexample, in polyethylene, —[—CH₂CH₂—]_(n)—, the repeat unit is —CH₂CH₂—.The subscript “n” denotes the degree of polymerization, that is, thenumber of repeat units linked together. When the value for “n” is leftundefined or where “n” is absent, it simply designates repetition of theformula within the brackets as well as the polymeric nature of thematerial. The concept of a repeat unit applies equally to where theconnectivity between the repeat units extends three dimensionally, suchas in metal organic frameworks, modified polymers, thermosettingpolymers, etc. Within the context of the dendrimer, the repeating unitmay also be described as the branching unit, interior layers, orgenerations. Similarly, the terminating group may also be described asthe surface group.

A “stereoisomer” or “optical isomer” is an isomer of a given compound inwhich the same atoms are bonded to the same other atoms, but where theconfiguration of those atoms in three dimensions differs. “Enantiomers”are stereoisomers of a given compound that are mirror images of eachother, like left and right hands. “Diastereomers” are stereoisomers of agiven compound that are not enantiomers. Chiral molecules contain achiral center, also referred to as a stereocenter or stereogenic center,which is any point, though not necessarily an atom, in a moleculebearing groups such that an interchanging of any two groups leads to astereoisomer. In organic compounds, the chiral center is typically acarbon, phosphorus or sulfur atom, though it is also possible for otheratoms to be stereocenters in organic and inorganic compounds. A moleculecan have multiple stereocenters, giving it many stereoisomers. Incompounds whose stereoisomerism is due to tetrahedral stereogeniccenters (e.g., tetrahedral carbon), the total number of hypotheticallypossible stereoisomers will not exceed 2^(n), where n is the number oftetrahedral stereocenters. Molecules with symmetry frequently have fewerthan the maximum possible number of stereoisomers. A 50:50 mixture ofenantiomers is referred to as a racemic mixture. Alternatively, amixture of enantiomers can be enantiomerically enriched so that oneenantiomer is present in an amount greater than 50%. Typically,enantiomers and/or diastereomers can be resolved or separated usingtechniques known in the art. It is contemplated that that for anystereocenter or axis of chirality for which stereochemistry has not beendefined, that stereocenter or axis of chirality can be present in its Rform, S form, or as a mixture of the R and S forms, including racemicand non-racemic mixtures. As used herein, the phrase “substantially freefrom other stereoisomers” means that the composition contains ≤15%, morepreferably ≤10%, even more preferably ≤5%, or most preferably ≤1% ofanother stereoisomer(s).

“Treatment” or “treating” includes (1) inhibiting a disease in a subjector patient experiencing or displaying the pathology or symptomatology ofthe disease (e.g., arresting further development of the pathology and/orsymptomatology), (2) ameliorating a disease in a subject or patient thatis experiencing or displaying the pathology or symptomatology of thedisease (e.g., reversing the pathology and/or symptomatology), and/or(3) effecting any measurable decrease in a disease in a subject orpatient that is experiencing or displaying the pathology orsymptomatology of the disease.

The above definitions supersede any conflicting definition in anyreference that is incorporated by reference herein. The fact thatcertain terms are defined, however, should not be considered asindicative that any term that is undefined is indefinite. Rather, allterms used are believed to describe the disclosure in terms such thatone of ordinary skill can appreciate the scope and practice the presentdisclosure.

B. CATIONIC IONIZABLE LIPIDS

In some aspects of the present disclosure, composition containingcompounds containing lipophilic and cationic components, wherein thecationic component is ionizable, are provided. In some embodiments, thecationic ionizable lipids contain one or more groups which is protonatedat physiological pH but may deprotonated at a pH above 8, 9, 10, 11, or12. The ionizable cationic group may contain one or more protonatableamines which are able to form a cationic group at physiological pH. Thecationic ionizable lipid compound may also further comprise one or morelipid components such as two or more fatty acids with C₆-C₂₄ alkyl oralkenyl carbon groups. These lipid groups may be attached through anester linkage or may be further added through a Michael addition to asulfur atom. In some embodiments, these compounds may be a dendrimer, adendron, a polymer, or a combination thereof.

In some embodiments, these cationic ionizable lipids are dendrimers,which are a polymer exhibiting regular dendritic branching, formed bythe sequential or generational addition of branched layers to or from acore and are characterized by a core, at least one interior branchedlayer, and a surface branched layer. (See Petar R. Dvornic and Donald A.Tomalia in Chem. in Britain, 641-645, August 1994.) In otherembodiments, the term “dendrimer” as used herein is intended to include,but is not limited to, a molecular architecture with an interior core,interior layers (or “generations”) of repeating units regularly attachedto this initiator core, and an exterior surface of terminal groupsattached to the outermost generation. A “dendron” is a species ofdendrimer having branches emanating from a focal point which is or canbe joined to a core, either directly or through a linking moiety to forma larger dendrimer. In some embodiments, the dendrimer structures haveradiating repeating groups from a central core which doubles with eachrepeating unit for each branch. In some embodiments, the dendrimersdescribed herein may be described as a small molecule, medium-sizedmolecules, lipids, or lipid-like material. These terms may be used todescribed compounds described herein which have a dendron likeappearance (e.g. molecules which radiate from a single focal point).

While dendrimers are polymers, dendrimers may be preferable totraditional polymers because they have a controllable structure, asingle molecular weight, numerous and controllable surfacefunctionalities, and traditionally adopt a globular conformation afterreaching a specific generation. Dendrimers can be prepared bysequentially reactions of each repeating unit to produce monodisperse,tree-like and/or generational structure polymeric structures. Individualdendrimers consist of a central core molecule, with a dendritic wedgeattached to one or more functional sites on that central core. Thedendrimeric surface layer can have a variety of functional groupsdisposed thereon including anionic, cationic, hydrophilic, or lipophilicgroups, according to the assembly monomers used during the preparation.

Modifying the functional groups and/or the chemical properties of thecore, repeating units, and the surface or terminating groups, theirphysical properties can be modulated. Some properties which can bevaried include, but are not limited to, solubility, toxicity,immunogenicity and bioattachment capability. Dendrimers are oftendescribed by their generation or number of repeating units in thebranches. A dendrimer consisting of only the core molecule is referredto as Generation 0, while each consecutive repeating unit along allbranches is Generation 1, Generation 2, and so on until the terminatingor surface group. In some embodiments, half generations are possibleresulting from only the first condensation reaction with the amine andnot the second condensation reaction with the thiol.

Preparation of dendrimers requires a level of synthetic control achievedthrough series of stepwise reactions comprising building the dendrimerby each consecutive group. Dendrimer synthesis can be of the convergentor divergent type. During divergent dendrimer synthesis, the molecule isassembled from the core to the periphery in a stepwise process involvingattaching one generation to the previous and then changing functionalgroups for the next stage of reaction. Functional group transformationis necessary to prevent uncontrolled polymerization. Such polymerizationwould lead to a highly branched molecule that is not monodisperse and isotherwise known as a hyperbranched polymer. Due to steric effects,continuing to react dendrimer repeat units leads to a sphere shaped orglobular molecule, until steric overcrowding prevents complete reactionat a specific generation and destroys the molecule's monodispersity.Thus, in some embodiments, the dendrimers of G1-G10 generation arespecifically contemplated. In some embodiments, the dendrimers comprise1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units, or any range derivabletherein. In some embodiments, the dendrimers used herein are G0, G1, G2,or G3. However, the number of possible generations (such as 11, 12, 13,14, 15, 20, or 25) may be increased by reducing the spacing units in thebranching polymer.

Additionally, dendrimers have two major chemical environments: theenvironment created by the specific surface groups on the terminationgeneration and the interior of the dendritic structure which due to thehigher order structure can be shielded from the bulk media and thesurface groups. Because of these different chemical environments,dendrimers have found numerous different potential uses including intherapeutic applications.

In some aspects, the dendrimers that may be used in the presentcompositions are assembled using the differential reactivity of theacrylate and methacrylate groups with amines and thiols. The dendrimersmay include secondary or tertiary amines and thioethers formed by thereaction of an acrylate group with a primary or secondary amine and amethacrylate with a mercapto group. Additionally, the repeating units ofthe dendrimers may contain groups which are degradable underphysiological conditions. In some embodiments, these repeating units maycontain one or more germinal diethers, esters, amides, or disulfidesgroups. In some embodiments, the core molecule is a monoamine whichallows dendritic polymerization in only one direction. In otherembodiments, the core molecule is a polyamine with multiple differentdendritic branches which each may comprise one or more repeating units.The dendrimer may be formed by removing one or more hydrogen atoms fromthis core. In some embodiments, these hydrogen atoms are on a heteroatomsuch as a nitrogen atom. In some embodiments, the terminating group is alipophilic groups such as a long chain alkyl or alkenyl group. In otherembodiments, the terminating group is a long chain haloalkyl orhaloalkenyl group. In other embodiments, the terminating group is analiphatic or aromatic group containing an ionizable group such as anamine (—NH₂) or a carboxylic acid (—CO₂H). In still other embodiments,the terminating group is an aliphatic or aromatic group containing oneor more hydrogen bond donors such as a hydroxide group, an amide group,or an ester.

The cationic ionizable lipids of the present disclosure may contain oneor more asymmetrically-substituted carbon or nitrogen atoms, and may beisolated in optically active or racemic form. Thus, all chiral,diastereomeric, racemic form, epimeric form, and all geometric isomericforms of a chemical formula are intended, unless the specificstereochemistry or isomeric form is specifically indicated. Cationicionizable lipids may occur as racemates and racemic mixtures, singleenantiomers, diastereomeric mixtures and individual diastereomers. Insome embodiments, a single diastereomer is obtained. The chiral centersof the cationic ionizable lipids of the present disclosure can have theS or the R configuration. Furthermore, it is contemplated that one ormore of the cationic ionizable lipids may be present as constitutionalisomers. In some embodiments, the compounds have the same formula butdifferent connectivity to the nitrogen atoms of the core. Withoutwishing to be bound by any theory, it is believed that such cationicionizable lipids exist because the starting monomers react first withthe primary amines and then statistically with any secondary aminespresent. Thus, the constitutional isomers may present the fully reactedprimary amines and then a mixture of reacted secondary amines.

Chemical formulas used to represent cationic ionizable lipids of thepresent disclosure will typically only show one of possibly severaldifferent tautomers. For example, many types of ketone groups are knownto exist in equilibrium with corresponding enol groups. Similarly, manytypes of imine groups exist in equilibrium with enamine groups.Regardless of which tautomer is depicted for a given formula, andregardless of which one is most prevalent, all tautomers of a givenchemical formula are intended.

The cationic ionizable lipids of the present disclosure may also havethe advantage that they may be more efficacious than, be less toxicthan, be longer acting than, be more potent than, produce fewer sideeffects than, be more easily absorbed than, and/or have a betterpharmacokinetic profile (e.g., higher oral bioavailability and/or lowerclearance) than, and/or have other useful pharmacological, physical, orchemical properties over, compounds known in the prior art, whether foruse in the indications stated herein or otherwise.

In addition, atoms making up the cationic ionizable lipids of thepresent disclosure are intended to include all isotopic forms of suchatoms. Isotopes, as used herein, include those atoms having the sameatomic number but different mass numbers. By way of general example andwithout limitation, isotopes of hydrogen include tritium and deuterium,and isotopes of carbon include ¹³C and ¹⁴C.

It should be recognized that the particular anion or cation forming apart of any salt form of a cationic ionizable lipids provided herein isnot critical, so long as the salt, as a whole, is pharmacologicallyacceptable. Additional examples of pharmaceutically acceptable salts andtheir methods of preparation and use are presented in Handbook ofPharmaceutical Salts: Properties, and Use (2002), which is incorporatedherein by reference.

In some embodiments, the ionizable cationic lipid is present in anamount from about from about 20 to about 23. In some embodiments, themolar percentage is from about 20, 20.5, 21, 21.5, 22, 22.5, to about 23or any range derivable therein. In other embodiments, the molarpercentage is from about 7.5 to about 20. In some embodiments, the molarpercentage is from about 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, to about 20 or any range derivable therein.

C. SELECTIVE ORGAN TARGETING (SORT) COMPOUND

In some aspects, the present disclosure comprise one or more selectiveorgan targeting (SORT) compound which leads to the selective delivery ofthe composition to a particular organ. This compound may be a lipid, asmall molecule therapeutic agent, a sugar, a vitamin, or a protein.

In some embodiments, the selective organ targeting (SORT) compound ispresent in the composition in a molar ratio from about 2%, 4%, 5%, 10%,15%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 45%, 50%,55%, 60%, 65%, to about 70%, or any range derivable therein. In someembodiments, the SORT compound may be present in an amount from about 5%to about 40%, from about 10% to about 40%, from about 20% to about 35%,from about 25% to about 35%, or from about 28% to about 34%.

In some embodiments, the SORT compound may be a lipid. A lipid is asmall molecule with two or more alkyl or alkenyl chains of C₆-C₂₄. Asmall molecule therapeutic agent is a compound containing less than 100non-hydrogen atoms and a weight of less than 2,000 Daltons. A sugar is amolecule comprising a molecular formula C_(n)H_(2n)O_(n), wherein n isfrom 3 to 7 or a combination of multiple molecules of that formula. Aprotein is a sequence of amino acids comprising at least 3 amino acidresidues. Proteins without a formal tertiary structure may also bereferred to as a peptide. The protein may also comprise an intactprotein with a tertiary structure. A vitamin is a macronutrient andconsists of one or more compounds selected from Vitamin A, Vitamin B₁,Vitamin B₂, Vitamin B₃, Vitamin B₅, Vitamin B₆, Vitamin B₇, Vitamin B₉,Vitamin B₁₂, Vitamin C, Vitamin D, Vitamin E, and Vitamin K.

1. Permanently Cationic Lipid

In some aspects, the present disclosure provides one or more lipids withone or more hydrophobic components and a permanently cationic group. Thepermanently cationic lipid may contain an group which has a positivecharge regardless of the pH. One permanently cationic group that may beused in the permanently cationic lipid is a quaternary ammonium group.These permanently cationic lipids include such structures as thosedescribed in the formula below:

wherein:

-   -   Y₁, Y₂, or Y₃ are each independently X₁C(O)R₁ or X₂N⁺R₃R₄R₅;        provided at least one of Y₁, Y₂, and Y₃ is X₂N⁺R₃R₄R₅;        -   R₁ is C₁-C₂₄ alkyl, C₁-C₂₄ substituted alkyl, C₁-C₂₄            alkenyl, C₁-C₂₄ substituted alkenyl;        -   X₁ is O or NR_(a), wherein Ra is hydrogen, C₁-C₄ alkyl, or            C₁-C₄ substituted alkyl;        -   X₂ is C₁-C₆ alkanediyl or C₁-C₆ substituted alkanediyl;        -   R₃, R₄, and R₅ are each independently C₁-C₂₄ alkyl, C₁-C₂₄            substituted alkyl, C₁-C₂₄ alkenyl, C₁-C₂₄ substituted            alkenyl;    -   A₁ is an anion with a charge equal to the number of X₂N⁺R₃R₄R₅        groups in the compound.

In another embodiment, the permanently cationic lipid is further definedby the formula:

wherein:

-   -   R₆-R₉ are each independently C₁-C₂₄ alkyl, C₁-C₂₄ substituted        alkyl, C₁-C₂₄ alkenyl, C₁-C₂₄ substituted alkenyl; provided at        least one of R₆-R₉ is a group of C₅-C₂₄; and    -   A₂ is a monovalent anion.

In another embodiments, the permanently cationic lipid is furtherdefined by the formula:

wherein:

-   -   R₁ and R₂ are each independently alkyl_((C8-C24)),        alkenyl_((C8-C24)), or a substituted version of either group;    -   R₃, R₃′, and R₃″ are each independently alkyl_((C≤6)) or        substituted alkyl_((C≤6));    -   R₄ is alkyl_((C≤6)) or substituted alkyl_((C≤6)); and    -   X⁻ is a monovalent anion.

In some embodiments, the permanently cationic lipid is present in anamount from about 4 to about 16 molar percentage of the total lipidcomposition. The composition may contain from about 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15 molar percentage, or any range derivable therein.In other embodiments, the composition may comprise from about 18 toabout 66 molar percentage of the total lipid composition. In someembodiments, the compositions may contain from about 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, or 66 molar percentage, or any range derivable therein.

2. Permanently Anionic Lipid

In some aspects, the present disclosure provides one or more lipids withone or more hydrophobic components and a permanently anionic group. Oneanionic group that may be used in the permanently anionic lipid is aphosphate group. The phosphate group may be a compound which isdeprotonated and possesses a negative charge at a pH below 8, 9, 10, 11,12, 13 or 14. The hydrophobic components may be one or more C₆-C₂₄ alkylor alkenyl groups. The compound may have one hydrophobic group, twohydrophobic groups, or three hydrophobic groups.

In some embodiments, the permanently anionic lipid has a structure ofthe formula:

wherein:

-   -   R₁ and R₂ are each independently alkyl_((C8-C24)),        alkenyl_((C8-C24)), or a substituted version of either group;    -   R₃ is hydrogen, alkyl_((C≤6)), or substituted alkyl_((C≤6)), or        —Y₁—R₄, wherein:        -   Y₁ is alkanediyl_((C≤6)) or substituted alkanediyl_((C≤6));            and        -   R₄ is acyloxy_((C≤8-24)) or substituted acyloxy_((C≤8-24)).

3. Phosphotidylcholine

In some aspects, the present disclosure provides one or more lipids withone or more hydrophobic components, a cationic amine group, and anegatively charged phosphate group. The cationic amine group may be aquaternary amine with three methyl groups attached to the nitrogen atom.The hydrophobic components may be one or more C₆-C₂₄ alkyl or alkenylgroups. The compound may have one hydrophobic group, two hydrophobicgroups, or three hydrophobic groups. In some embodiments, thephophotidylcholine compound is further defined as:

wherein:

-   -   R₁ and R₂ are each independently alkyl_((C8-C24)),        alkenyl_((C8-C24)), or a substituted version of either group;    -   R₃, R₃′, and R₃″ are each independently alkyl_((C≤6)) or        substituted alkyl_((C≤6)); and    -   X⁻ is a monovalent anion.

D. ADDITIONAL LIPIDS IN THE LIPID NANOPARTICLES

In some aspects of the present disclosure, compositions containing oneor more lipids are mixed with the cationic ionizable lipids to create acomposition. In some embodiments, the cationic ionizable lipids aremixed with 1, 2, 3, 4, or 5 different types of lipids. It iscontemplated that the cationic ionizable lipids can be mixed withmultiple different lipids of a single type. In some embodiments, thecationic ionizable lipids compositions comprise at least a steroid or asteroid derivative, a PEG lipid, and a phospholipid.

In some embodiments, the lipid nanoparticles are preferentiallydelivered to a target organ. In some embodiments, the target organ isselected from the lungs, the heart, the brain, the spleen, the bonemarrow, the bones, the skeletal muscles, the stomach, the smallintestine, the large intestine, the kidneys, the bladder, the breast,the liver, the testes, the ovaries, the uterus, the spleen, the thymus,the brainstem, the cerebellum, the spinal cord, the eye, the ear, thetongue, or the skin. Alternatively, the composition may bepreferentially delivered to a target organ system such as the nervoussystem, the cardiovascular system, or the respiratory system or a partof one of these organ system. As used herein, the term “preferentiallydelivered” is used to refer to a composition which is delivered to thetarget organ or organ system in at least 25% of the amount administered.This term is used to refer to a composition in which at least 25%, 50%,or at least 75% of the amount administered.

1. Steroids and Steroid Derivatives

In some aspects of the present disclosure, the cationic ionizable lipidsare mixed with one or more steroid or a steroid derivative to create acomposition. In some embodiments, the steroid or steroid derivativecomprises any steroid or steroid derivative. As used herein, in someembodiments, the term “steroid” is a class of compounds with a four ring17 carbon cyclic structure which can further comprises one or moresubstitutions including alkyl groups, alkoxy groups, hydroxy groups, oxogroups, acyl groups, or a double bond between two or more carbon atoms.In one aspect, the ring structure of a steroid comprises three fusedcyclohexyl rings and a fused cyclopentyl ring as shown in the formulabelow:

In some embodiments, a steroid derivative comprises the ring structureabove with one or more non-alkyl substitutions. In some embodiments, thesteroid or steroid derivative is a sterol wherein the formula is furtherdefined as:

In some embodiments of the present disclosure, the steroid or steroidderivative is a cholestane or cholestane derivative. In a cholestane,the ring structure is further defined by the formula:

As described above, a cholestane derivative includes one or morenon-alkyl substitution of the above ring system. In some embodiments,the cholestane or cholestane derivative is a cholestene or cholestenederivative or a sterol or a sterol derivative. In other embodiments, thecholestane or cholestane derivative is both a cholestere and a sterol ora derivative thereof.

In some embodiments, the compositions may further comprise a molarpercentage of the steroid to the total lipid composition from about 40to about 46. In some embodiments, the molar percentage is from about 40,41, 42, 43, 44, 45, to about 46 or any range derivable therein. In otherembodiments, the molar percentage of the steroid relative to the totallipid composition is from about 15 to about 40. In some embodiments, themolar percentage is 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,or 40, or any range derivable therein.

2. PEG or PEGylated Lipid

In some aspects of the present disclosure, the polymers are mixed withone or more PEGylated lipids (or PEG lipid) to create a lipidcomposition. In some embodiments, the present disclosure comprises usingany lipid to which a PEG group has been attached. In some embodiments,the PEG lipid is a diglyceride which also comprises a PEG chain attachedto the glycerol group. In other embodiments, the PEG lipid is a compoundwhich contains one or more C₆-C₂₄ long chain alkyl or alkenyl group or aC₆-C₂₄ fatty acid group attached to a linker group with a PEG chain.Some non-limiting examples of a PEG lipid includes a PEG modifiedphosphatidylethanolamine and phosphatidic acid, a PEG ceramideconjugated, PEG modified dialkylamines and PEG modified1,2-diacyloxypropan-3-amines, PEG modified diacylglycerols anddialkylglycerols. In some embodiments, PEG modifieddiastearoylphosphatidylethanolamine or PEG modifieddimyristoyl-sn-glycerol. In some embodiments, the PEG modification ismeasured by the molecular weight of PEG component of the lipid. In someembodiments, the PEG modification has a molecular weight from about 100to about 15,000. In some embodiments, the molecular weight is from about200 to about 500, from about 400 to about 5,000, from about 500 to about3,000, or from about 1,200 to about 3,000. The molecular weight of thePEG modification is from about 100, 200, 400, 500, 600, 800, 1,000,1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000,4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500, to about15,000. Some non-limiting examples of lipids that may be used in thepresent disclosure are taught by U.S. Pat. No. 5,820,873, WO2010/141069, or U.S. Pat. No. 8,450,298, which is incorporated herein byreference.

In another aspect, the PEG lipid has the formula:

wherein: R₁₂ and R₁₃ are each independently alkyl_((C≤24)),alkenyl_((C≤24)), or a substituted version of either of these groups;R_(e) is hydrogen, alkyl_((C≤8)), or substituted alkyl_((C≤8)); and x is1-250. In some embodiments, R_(e) is alkyl_((C≤8)) such as methyl. R₁₂and R₁₃ are each independently alkyl_((C≤4-20)). In some embodiments, xis 5-250. In one embodiment, x is 5-125 or x is 100-250. In someembodiments, the PEG lipid is 1,2-dimyristoyl-sn-glycerol,methoxypolyethylene glycol.

In another aspect, the PEG lipid has the formula:

wherein: n₁ is an integer between 1 and 100 and n₂ and n₃ are eachindependently selected from an integer between 1 and 29. In someembodiments, n₁ is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, or 100, or any range derivable therein. In someembodiments, n₁ is from about 30 to about 50. In some embodiments, n₂ isfrom 5 to 23. In some embodiments, n₂ is 11 to about 17. In someembodiments, n₃ is from 5 to 23. In some embodiments, n₃ is 11 to about17.

In some embodiments, the compositions may further comprise a molarpercentage of the PEG lipid to the total lipid composition from about4.0 to about 4.6. In some embodiments, the molar percentage is fromabout 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, to about 4.6 or any range derivabletherein. In other embodiments, the molar percentage is from about 1.5 toabout 4.0. In some embodiments, the molar percentage is from about 1.5,1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, to about 4.0 or any rangederivable therein.

3. Phospholipid

In some aspects of the present disclosure, the polymers are mixed withone or more phospholipids to create a composition. In some embodiments,any lipid which also comprises a phosphate group. In some embodiments,the phospholipid is a structure which contains one or two long chainC₆-C₂₄ alkyl or alkenyl groups, a glycerol or a sphingosine, one or twophosphate groups, and, optionally, a small organic molecule. In someembodiments, the small organic molecule is an amino acid, a sugar, or anamino substituted alkoxy group, such as choline or ethanolamine. In someembodiments, the phospholipid is a phosphatidylcholine. In someembodiments, the phospholipid is distearoylphosphatidylcholine ordioleoylphosphatidylethanolamine.

In some embodiments, the compositions may further comprise a molarpercentage of the phospholipid to the total lipid composition from about20 to about 23. In some embodiments, the molar percentage is from about20, 20.5, 21, 21.5, 22, 22.5, to about 23 or any range derivabletherein. In other embodiments, the molar percentage is from about 7.5 toabout 20. In some embodiments, the molar percentage is from about 7.5,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, to about 20 or any rangederivable therein.

E. THERAPEUTIC AGENTS

1. Nucleic Acids

In some aspects of the present disclosure, the lipid compositionscomprise one or more nucleic acids. In some embodiments, the lipidcomposition comprises one or more nucleic acids present in a weightratio to the lipid composition from about 5:1 to about 1:100. In someembodiments, the weight ratio of nucleic acid to lipid composition isfrom about 5:1, 2.5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35,1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100, or any rangederivable therein. In some embodiments, the weight ratio is about 1:40.In addition, it should be clear that the present disclosure is notlimited to the specific nucleic acids disclosed herein. The presentdisclosure is not limited in scope to any particular source, sequence,or type of nucleic acid, however, as one of ordinary skill in the artcould readily identify related homologs in various other sources of thenucleic acid including nucleic acids from non-human species (e.g.,mouse, rat, rabbit, dog, monkey, gibbon, chimp, ape, baboon, cow, pig,horse, sheep, cat and other species). It is contemplated that thenucleic acid used in the present disclosure can comprises a sequencebased upon a naturally-occurring sequence. Allowing for the degeneracyof the genetic code, sequences that have at least about 50%, usually atleast about 60%, more usually about 70%, most usually about 80%,preferably at least about 90% and most preferably about 95% ofnucleotides that are identical to the nucleotide sequence of thenaturally-occurring sequence. In another embodiment, the nucleic acid isa complementary sequence to a naturally occurring sequence, orcomplementary to 75%, 80%, 85%, 90%, 95% and 100%. Longerpolynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 orlonger are contemplated herein.

The nucleic acid used herein may be derived from genomic DNA, i.e.,cloned directly from the genome of a particular organism. In preferredembodiments, however, the nucleic acid would comprise complementary DNA(cDNA). Also contemplated is a cDNA plus a natural intron or an intronderived from another gene; such engineered molecules are sometimereferred to as “mini-genes.” At a minimum, these and other nucleic acidsof the present disclosure may be used as molecular weight standards in,for example, gel electrophoresis.

The term “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA primarily contains coding sequences of thecorresponding protein. There may be times when the full or partialgenomic sequence is preferred, such as where the non-coding regions arerequired for optimal expression or where non-coding regions such asintrons are to be targeted in an antisense strategy.

In some embodiments, the nucleic acid comprises one or more antisensesegments which inhibits expression of a gene or gene product. Antisensemethodology takes advantage of the fact that nucleic acids tend to pairwith “complementary” sequences. By complementary, it is meant thatpolynucleotides are those which are capable of base-pairing according tothe standard Watson-Crick complementarity rules. That is, the largerpurines will base pair with the smaller pyrimidines to form combinationsof guanine paired with cytosine (G:C) and adenine paired with eitherthymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) inthe case of RNA. Inclusion of less common bases such as inosine,5-methylcytosine, 6-methyladenine, hypoxanthine and others inhybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads totriple-helix formation; targeting RNA will lead to double-helixformation. Antisense polynucleotides, when introduced into a targetcell, specifically bind to their target polynucleotide and interferewith transcription, RNA processing, transport, translation and/orstability. Antisense RNA constructs, or DNA encoding such antisenseRNA's, may be employed to inhibit gene transcription or translation orboth within a host cell, either in vitro or in vivo, such as within ahost animal, including a human subject.

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. It is contemplated that the most effective antisense constructswill include regions complementary to intron/exon splice junctions.Thus, it is proposed that a preferred embodiment includes an antisenseconstruct with complementarity to regions within 50-200 bases of anintron-exon splice junction. It has been observed that some exonsequences can be included in the construct without seriously affectingthe target selectivity thereof. The amount of exonic material includedwill vary depending on the particular exon and intron sequences used.One can readily test whether too much exon DNA is included simply bytesting the constructs in vitro to determine whether normal cellularfunction is affected or whether the expression of related genes havingcomplementary sequences is affected.

As stated above, “complementary” or “antisense” means polynucleotidesequences that are substantially complementary over their entire lengthand have very few base mismatches. For example, sequences of fifteenbases in length may be termed complementary when they have complementarynucleotides at thirteen or fourteen positions. Naturally, sequenceswhich are completely complementary will be sequences which are entirelycomplementary throughout their entire length and have no basemismatches. Other sequences with lower degrees of homology also arecontemplated. For example, an antisense construct which has limitedregions of high homology, but also contains a non-homologous region(e.g., ribozyme; see below) could be designed. These molecules, thoughhaving less than 50% homology, would bind to target sequences underappropriate conditions.

2. Modified Nucleobases

In some embodiments, the nucleic acids of the present disclosurecomprise one or more modified nucleosides comprising a modified sugarmoiety. Such compounds comprising one or more sugar-modified nucleosidesmay have desirable properties, such as enhanced nuclease stability orincreased binding affinity with a target nucleic acid relative to anoligonucleotide comprising only nucleosides comprising naturallyoccurring sugar moieties. In some embodiments, modified sugar moietiesare substituted sugar moieties. In some embodiments, modified sugarmoieties are sugar surrogates. Such sugar surrogates may comprise one ormore substitutions corresponding to those of substituted sugar moieties.

In some embodiments, modified sugar moieties are substituted sugarmoieties comprising one or more non-bridging sugar substituent,including but not limited to substituents at the 2′ and/or 5′ positions.Examples of sugar substituents suitable for the 2′-position, include,but are not limited to: 2′-F, 2′-OCH₃ (“OMe” or “O-methyl”), and2′-O(CH₂)₂OCH₃ (“MOE”). In certain embodiments, sugar substituents atthe 2′ position is selected from allyl, amino, azido, thio, O-allyl,O—C₁-C₁₀ alkyl, O—C₁-C₁₀ substituted alkyl; OCF₃, O(CH₂)₂SCH₃,O(CH₂)₂—O—N(Rm)(Rn), and O—CH₂—C(═O)—N(Rm)(Rn), where each Rm and Rn is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl. Examplesof sugar substituents at the 5′-position, include, but are not limitedto: 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy. In some embodiments,substituted sugars comprise more than one non-bridging sugarsubstituent, for example, T-F-5′-methyl sugar moieties (see, e.g., PCTInternational Application WO 2008/101157, for additional 5′,2′-bissubstituted sugar moieties and nucleosides).

Nucleosides comprising 2′-substituted sugar moieties are referred to as2′-substituted nucleosides. In some embodiments, a 2′-substitutednucleoside comprises a 2′-substituent group selected from halo, allyl,amino, azido, SH, CN, OCN, CF₃, OCF₃, O, S, or N(Rm)-alkyl; O, S, orN(Rm)-alkenyl; O, S or N(Rm)-alkynyl; O-alkylenyl-O-alkyl, alkynyl,alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃,O(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R_(n) is, independently, H, an amino protecting group orsubstituted or unsubstituted C₁-C₁₀ alkyl. These 2′-substituent groupscan be further substituted with one or more substituent groupsindependently selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro (NO₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl,alkenyl and alkynyl.

In some embodiments, a 2′-substituted nucleoside comprises a2′-substituent group selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂,CH₂—CH═CH₂, O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

In some embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, OCF₃, O—CH₃,OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N(CH₃)₂, andO—CH₂—C(═O)—N(H)CH₃.

In some embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, O—CH₃, andOCH₂CH₂OCH₃.

Certain modified sugar moieties comprise a bridging sugar substituentthat forms a second ring resulting in a bicyclic sugar moiety. In somesuch embodiments, the bicyclic sugar moiety comprises a bridge betweenthe 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′ sugarsubstituents, include, but are not limited to: —[C(R_(a))(R_(b))]_(n)—,—[C(R_(a))(R_(b))]_(n)—O—, —C(R_(a)R_(b))—N(R)—O— or,—C(R_(a)R_(b))—O—N(R)—; 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)—O-2′ (LNA);4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (cEt) and4′-CH(CH₂OCH₃)—O-2′, and analogs thereof (see, e.g., U.S. Pat. No.7,399,845); 4′-C(CH₃)(CH₃)—O-2′ and analogs thereof, (see, e.g., WO2009/006478); 4′-CH₂—N(OCH₃)-2′ and analogs thereof (see, e.g.,WO2008/150729); 4′-CH₂—O—N(CH₃)-2′ (see, e.g., US2004/0171570, publishedSep. 2, 2004); 4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)—O-2′-, wherein each Ris, independently, H, a protecting group, or C₁-C₁₂ alkyl;4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂ alkyl, or a protecting group(see, U.S. Pat. No. 7,427,672); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g.,Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and4′-CH₂—C(═CH₂)-2′ and analogs thereof (see, PCT InternationalApplication WO 2008/154401).

In some embodiments, such 4′ to 2′ bridges independently comprise from 1to 4 linked groups independently selected from —[C(R_(a))(R_(b))]_(n)—,—C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—,—Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—; wherein:

-   -   x is 0, 1, or 2;    -   n is 1, 2, 3, or 4;    -   each R_(a) and R_(b) is, independently, H, a protecting group,        hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂        alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted        C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl,        heterocycle radical, substituted heterocycle radical,        heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,        substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁,        N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl        (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and    -   each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted        C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂        alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted        C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycle        radical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl,        substituted C₁-C₁₂ aminoalkyl, or a protecting group.

Nucleosides comprising bicyclic sugar moieties are referred to asbicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are notlimited to, (A) α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-Methyleneoxy(4′-CH₂—O-2′) BNA (also referred to as locked nucleic acid or LNA), (C)Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) Aminooxy (4′-CH₂—O—N(R)-2′) BNA,(E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F) Methyl(methyleneoxy)(4′-CH(CH₃)—O-2′) BNA (also referred to as constrained ethyl or cEt),(G) methylene-thio (4′-CH₂—S-2′) BNA, (H) methylene-amino(4′-CH₂—N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA,(J) propylene carbocyclic (4′-(CH₂)3-2′) BNA, and (K)Methoxy(ethyleneoxy) (4′-CH(CH₂OMe)-O-2′) BNA (also referred to asconstrained MOE or cMOE).

Additional bicyclic sugar moieties are known in the art, for example:Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al.,Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad.Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem.Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63,10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379(Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2,5561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr.Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 7,053,207,6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and7,399,845; WO 2004/106356, WO 1994/14226, WO 2005/021570, and WO2007/134181; U.S. Patent Publication Nos. US 2004/0171570, US2007/0287831, and US 2008/0039618; U.S. Ser. Nos. 12/129,154,60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787,and 61/099,844; and PCT International Applications Nos.PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922.

In some embodiments, bicyclic sugar moieties and nucleosidesincorporating such bicyclic sugar moieties are further defined byisomeric configuration. For example, a nucleoside comprising a 4′-2′methylene-oxy bridge, may be in the .alpha.-L configuration or in the.beta.-D configuration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′)bicyclic nucleosides have been incorporated into antisenseoligonucleotides that showed antisense activity (Frieden et al., NucleicAcids Research, 2003, 21, 6365-6372).

In some embodiments, substituted sugar moieties comprise one or morenon-bridging sugar substituent and one or more bridging sugarsubstituent (e.g., 5′-substituted and 4′-2′ bridged sugars; PCTInternational Application WO 2007/134181, wherein LNA is substitutedwith, for example, a 5′-methyl or a 5′-vinyl group).

In some embodiments, modified sugar moieties are sugar surrogates. Insome such embodiments, the oxygen atom of the naturally occurring sugaris substituted, e.g., with a sulfer, carbon or nitrogen atom. In somesuch embodiments, such modified sugar moiety also comprises bridgingand/or non-bridging substituents as described above. For example,certain sugar surrogates comprise a 4′-sulfur atom and a substitution atthe 2′-position (see, e.g., published U.S. Patent Application US2005/0130923) and/or the 5′ position. By way of additional example,carbocyclic bicyclic nucleosides having a 4′-2′ bridge have beendescribed (see, e.g., Freier et al., Nucleic Acids Research, 1997,25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71,7731-7740).

In some embodiments, sugar surrogates comprise rings having other than5-atoms. For example, in some embodiments, a sugar surrogate comprises asix-membered tetrahydropyran. Such tetrahydropyrans may be furthermodified or substituted. Nucleosides comprising such modifiedtetrahydropyrans include, but are not limited to, hexitol nucleic acid(HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (seeLeumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), and fluoro HNA(F-HNA).

In some embodiments, the modified THP nucleosides of Formula VII areprovided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other thanH. In some embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ ismethyl. In some embodiments, THP nucleosides of Formula VII are providedwherein one of R₁ and R₂ is F. In certain embodiments, R₁ is fluoro andR₂ is H, R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxy and R₂ is H.

Many other bicyclo and tricyclo sugar surrogate ring systems are alsoknown in the art that can be used to modify nucleosides forincorporation into antisense compounds (see, e.g., review article:Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 for other disclosed 5′,2′-bis substitutednucleosides) and replacement of the ribosyl ring oxygen atom with S andfurther substitution at the 2′-position (see U.S. Patent Publication US2005/0130923) or alternatively 5′-substitution of a bicyclic nucleicacid (see PCT International Application WO 2007/134181 wherein a4′-CH₂—O-2′ bicyclic nucleoside is further substituted at the 5′position with a 5′-methyl or a 5′-vinyl group). The synthesis andpreparation of carbocyclic bicyclic nucleosides along with theiroligomerization and biochemical studies have also been described (see,e.g., Srivastava et al., 2007).

In some embodiments, the present disclosure provides oligonucleotidescomprising modified nucleosides. Those modified nucleotides may includemodified sugars, modified nucleobases, and/or modified linkages. Thespecific modifications are selected such that the resultingoligonucleotides possess desirable characteristics. In some embodiments,oligonucleotides comprise one or more RNA-like nucleosides. In someembodiments, oligonucleotides comprise one or more DNA-like nucleotides.

In some embodiments, nucleosides of the present disclosure comprise oneor more unmodified nucleobases. In certain embodiments, nucleosides ofthe present disclosure comprise one or more modified nucleobases.

In some embodiments, modified nucleobases are selected from: universalbases, hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases as defined herein. 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine;5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynylCH₃) uracil and cytosine and other alkynyl derivatives of pyrimidinebases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine,3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases,promiscuous bases, size-expanded bases, and fluorinated bases as definedherein. Further modified nucleobases include tricyclic pyrimidines suchas phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.,9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazin-2(3H)-one),carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindolecytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modifiednucleobases may also include those in which the purine or pyrimidinebase is replaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz,J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed byEnglisch et al., 1991; and those disclosed by Sanghvi, Y. S., 1993.

Representative United States Patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include without limitation, U.S. Pat. Nos.3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985;5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, each of whichis herein incorporated by reference in its entirety.

In some embodiments, the present disclosure provides oligonucleotidescomprising linked nucleosides. In such embodiments, nucleosides may belinked together using any internucleoside linkage. The two main classesof internucleoside linking groups are defined by the presence or absenceof a phosphorus atom. Representative phosphorus containinginternucleoside linkages include, but are not limited to,phosphodiesters (P═O), phosphotriesters, methylphosphonates,phosphoramidate, and phosphorothioates (P═S). Representativenon-phosphorus containing internucleoside linking groups include, butare not limited to, methylenemethylimino (—CH₂—N(CH₃)—O—CH₂—),thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane(—O—Si(H)₂—O—); and N,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—).Modified linkages, compared to natural phosphodiester linkages, can beused to alter, typically increase, nuclease resistance of theoligonucleotide. In some embodiments, internucleoside linkages having achiral atom can be prepared as a racemic mixture, or as separateenantiomers. Representative chiral linkages include, but are not limitedto, alkylphosphonates and phosphorothioates. Methods of preparation ofphosphorous-containing and non-phosphorous-containing internucleosidelinkages are well known to those skilled in the art.

The oligonucleotides described herein contain one or more asymmetriccenters and thus give rise to enantiomers, diastereomers, and otherstereoisomeric configurations that may be defined, in terms of absolutestereochemistry, as (R) or (S), α or β such as for sugar anomers, or as(D) or (L) such as for amino acids etc. Included in the antisensecompounds provided herein are all such possible isomers, as well astheir racemic and optically pure forms.

Neutral internucleoside linkages include without limitation,phosphotriesters, methylphosphonates, MMI (3′-CH₂—N(CH₃)—O-5′), amide-3(3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal(3′-O—CH₂—O-5′), and thioformacetal (3′-S—CH₂—O-5′). Further neutralinternucleoside linkages include nonionic linkages comprising siloxane(dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonateester and amides (See for example: Carbohydrate Modifications inAntisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS SymposiumSeries 580; Chapters 3 and 4, 40-65). Further neutral internucleosidelinkages include nonionic linkages comprising mixed N, O, S and CH₂component parts.

Additional modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide and the 5′ position of 5′ terminal nucleotide. Forexample, one additional modification of the ligand conjugatedoligonucleotides of the present disclosure involves chemically linkingto the oligonucleotide one or more additional non-ligand moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. Such moieties include but are not limitedto lipid moieties such as a cholesterol moiety (Letsinger et al., 1989),cholic acid (Manoharan et al., 1994), a thioether, e.g.,hexyl-5-tritylthiol (Manoharan et al., 1992; Manoharan et al., 1993), athiocholesterol (Oberhauser et al., 1992), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., 1991; Kabanovet al., 1990; Svinarchuk et al., 1993), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., 1995;Shea et al., 1990), a polyamine or a polyethylene glycol chain(Manoharan et al., 1995), or adamantane acetic acid (Manoharan et al.,1995), a palmityl moiety (Mishra et al., 1995), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., 1996).

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis herein incorporated by reference.

3. Proteins

In some embodiments, the compositions may further comprise one or moreproteins. Some proteins may include enzymes such as nuclease enzymes.The compositions described herein may comprise one or more CRISPRassociated proteins (e.g. CRISPR enzyme) including a Cas protein.Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3,Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3,Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4,homologs thereof, or modified versions thereof. These enzymes are known;for example, the amino acid sequence of S. pyogenes Cas9 protein may befound in the SwissProt database under accession number Q99ZW2.

The protein in the compositions described herein may be Cas9 (e.g., fromS. pyogenes or S. pneumonia). The CRISPR enzyme can direct cleavage ofone or both strands at the location of a target sequence, such as withinthe target sequence and/or within the complement of the target sequence.The CRISPR enzyme may be mutated with respect to a correspondingwild-type enzyme such that the mutated CRISPR enzyme lacks the abilityto cleave one or both strands of a target polynucleotide containing atarget sequence. For example, an aspartate-to-alanine substitution(D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes convertsCas9 from a nuclease that cleaves both strands to a nickase (cleaves asingle strand). In some embodiments, a Cas9 nickase may be used incombination with guide sequence(s), e.g., two guide sequences, whichtarget respectively sense and antisense strands of the DNA target. Thiscombination allows both strands to be nicked and used to induce NHEJ orHDR.

In some embodiments, the present disclosure provides compoundscontaining one or more therapeutic proteins. The therapeutic proteinsthat may be included in the composition include a wide range ofmolecules such as cytokines, chemokines, interleukins, interferons,growth factors, coagulation factors, anti-coagulants, blood factors,bone morphogenic proteins, immunoglobulins, and enzymes. Somenon-limiting examples of particular therapeutic proteins includeErythropoietin (EPO), Granulocyte colony-stimulating factor (G-CSF),Alpha-galactosidase A, Alpha-L-iduronidase, Thyrotropin α,N-acetylgalactosamine-4-sulfatase (rhASB), Dornase alfa, Tissueplasminogen activator (TPA) Activase, Glucocerebrosidase, Interferon(IF) β-1a, Interferon β-1b, Interferon γ, Interferon α, TNF-α, IL-1through IL-36, Human growth hormone (rHGH), Human insulin (BHI), Humanchorionic gonadotropin α, Darbepoetin α, Follicle-stimulating hormone(FSH), and Factor VIII.

4. Small Molecule Therapeutic Agents

In some aspects, the present disclosure provides compositions comprisinga therapeutic agent. The therapeutic agent may be a small molecule suchas a 7-Methoxypteridine, 7-Methylpteridine, abacavir, abafungin,abarelix, acebutolol, acenaphthene, acetaminophen, acetanilide,acetazolamide, acetohexamide, acetretin, acrivastine, adenine,adenosine, alatrofloxacin, albendazole, albuterol, alclofenac,aldesleukin, alemtuzumab, alfuzosin, alitretinoin, allobarbital,allopurinol, all-transretinoic acid (ATRA), aloxiprin, alprazolam,alprenolol, altretamine, amifostine, amiloride, aminoglutethimide,aminopyrine, amiodarone HCl, amitriptyline, amlodipine, amobarbital,amodiaquine, amoxapine, amphetamine, amphotericin, amphotericin B,ampicillin, amprenavir, amsacrine, amylnitrate, amylobarbitone,anastrozole, anrinone, anthracene, anthracyclines, aprobarbital, arsenictrioxide, asparaginase, aspirin, astemizole, atenolol, atorvastatin,atovaquone, atrazine, atropine, atropine azathioprine, auranofin,azacitidine, azapropazone, azathioprine, azintamide, azithromycin,aztreonum, baclofen, barbitone, BCG live, beclamide, beclomethasone,bendroflumethiazide, benezepril, benidipine, benorylate, benperidol,bentazepam, benzamide, benzanthracene, benzathine penicillin, benzhexolHCl, benznidazole, benzodiazepines, benzoic acid, bepheniumhydroxynaphthoate, betamethasone, bevacizumab (avastin), bexarotene,bezafibrate, bicalutamide, bifonazole, biperiden, bisacodyl, bisantrene,bleomycin, bleomycin, bortezomib, brinzolamide, bromazepam,bromocriptine mesylate, bromperidol, brotizolam, budesonide, bumetanide,bupropion, busulfan, butalbital, butamben, butenafine HCl,butobarbitone, butobarbitone (butethal), butoconazole, butoconazolenitrate, butylparaben, caffeine, calcifediol, calciprotriene,calcitriol, calusterone, cambendazole, camphor, camptothecin,camptothecin analogs, candesartan, capecitabine, capsaicin, captopril,carbamazepine, carbimazole, carbofuran, carboplatin, carbromal,carimazole, carmustine, cefamandole, cefazolin, cefixime, ceftazidime,cefuroxime axetil, celecoxib, cephradine, cerivastatin, cetrizine,cetuximab, chlorambucil, chloramphenicol, chlordiazepoxide,chlormethiazole, chloroquine, chlorothiazide, chlorpheniramine,chlorproguanil HCl, chlorpromazine, chlorpropamide, chlorprothixene,chlorpyrifos, chlortetracycline, chlorthalidone, chlorzoxazone,cholecalciferol, chrysene, cilostazol, cimetidine, cinnarizine,cinoxacin, ciprofibrate, ciprofloxacin HCl, cisapride, cisplatin,citalopram, cladribine, clarithromycin, clemastine fumarate, clioquinol,clobazam, clofarabine, clofazimine, clofibrate, clomiphene citrate,clomipramine, clonazepam, clopidogrel, clotiazepam, clotrimazole,clotrimazole, cloxacillin, clozapine, cocaine, codeine, colchicine,colistin, conjugated estrogens, corticosterone, cortisone, cortisoneacetate, cyclizine, cyclobarbital, cyclobenzaprine,cyclobutane-spirobarbiturate, cycloethane-spirobarbiturate,cycloheptane-spirobarbiturate, cyclohexane-spirobarbiturate,cyclopentane-spirobarbiturate, cyclophosphamide,cyclopropane-spirobarbiturate, cycloserine, cyclosporin, cyproheptadine,cyproheptadine HCl, cytarabine, cytosine, dacarbazine, dactinomycin,danazol, danthron, dantrolene sodium, dapsone, darbepoetin alfa,darodipine, daunorubicin, decoquinate, dehydroepiandrosterone,delavirdine, demeclocycline, denileukin, deoxycorticosterone,desoxymethasone, dexamethasone, dexamphetamine, dexchlorpheniramine,dexfenfluramine, dexrazoxane, dextropropoxyphene, diamorphine,diatrizoicacid, diazepam, diazoxide, dichlorophen, dichlorprop,diclofenac, dicumarol, didanosine, diflunisal, digitoxin, digoxin,dihydrocodeine, dihydroequilin, dihydroergotamine mesylate,diiodohydroxyquinoline, diltiazem HCl, diloxamide furoate,dimenhydrinate, dimorpholamine, dinitolmide, diosgenin, diphenoxylateHCl, diphenyl, dipyridamole, dirithromycin, disopyramide, disulfiram,diuron, docetaxel, domperidone, donepezil, doxazosin, doxazosin HCl,doxorubicin (neutral), doxorubicin HCl, doxycycline, dromostanolonepropionate, droperidol, dyphylline, echinocandins, econazole, econazolenitrate, efavirenz, ellipticine, enalapril, enlimomab, enoximone,epinephrine, epipodophyllotoxin derivatives, epirubicin, epoetinalfa,eposartan, equilenin, equilin, ergocalciferol, ergotamine tartrate,erlotinib, erythromycin, estradiol, estramustine, estriol, estrone,ethacrynic acid, ethambutol, ethinamate, ethionamide, ethopropazine HCl,ethyl-4-aminobenzoate (benzocaine), ethylparaben, ethinylestradiol,etodolac, etomidate, etoposide, etretinate, exemestane, felbamate,felodipine, fenbendazole, fenbuconazole, fenbufen, fenchlorphos,fenclofenac, fenfluramine, fenofibrate, fenoldepam, fenoprofen calcium,fenoxycarb, fenpiclonil, fentanyl, fenticonazole, fexofenadine,filgrastim, finasteride, flecamide acetate, floxuridine, fludarabine,fluconazole, fluconazole, flucytosine, fludioxonil, fludrocortisone,fludrocortisone acetate, flufenamic acid, flunanisone, flunarizine HCl,flunisolide, flunitrazepam, fluocortolone, fluometuron, fluorene,fluorouracil, fluoxetine HCl, fluoxymesterone, flupenthixol decanoate,fluphenthixol decanoate, flurazepam, flurbiprofen, fluticasonepropionate, fluvastatin, folic acid, fosenopril, fosphenytoin sodium,frovatriptan, furosemide, fulvestrant, furazolidone, gabapentin, G-BHC(Lindane), gefitinib, gemcitabine, gemfibrozil, gemtuzumab, glafenine,glibenclamide, gliclazide, glimepiride, glipizide, glutethimide,glyburide, Glyceryltrinitrate (nitroglycerin), goserelin acetate,grepafloxacin, griseofulvin, guaifenesin, guanabenz acetate, guanine,halofantrine HCl, haloperidol, hydrochlorothiazide, heptabarbital,heroin, hesperetin, hexachlorobenzene, hexethal, histrelin acetate,hydrocortisone, hydroflumethiazide, hydroxyurea, hyoscyamine,hypoxanthine, ibritumomab, ibuprofen, idarubicin, idobutal, ifosfamide,ihydroequilenin, imatinib mesylate, imipenem, indapamide, indinavir,indomethacin, indoprofen, interferon alfa-2a, interferon alfa-2b,iodamide, iopanoic acid, iprodione, irbesartan, irinotecan,isavuconazole, isocarboxazid, isoconazole, isoguanine, isoniazid,isopropylbarbiturate, isoproturon, isosorbide dinitrate, isosorbidemononitrate, isradipine, itraconazole, itraconazole, itraconazole(Itra), ivermectin, ketoconazole, ketoprofen, ketorolac, khellin,labetalol, lamivudine, lamotrigine, lanatoside C, lanosprazole, L-DOPA,leflunomide, lenalidomide, letrozole, leucovorin, leuprolide acetate,levamisole, levofloxacin, lidocaine, linuron, lisinopril, lomefloxacin,lomustine, loperamide, loratadine, lorazepam, lorefloxacin,lormetazepam, losartan mesylate, lovastatin, lysuride maleate,Maprotiline HCl, mazindol, mebendazole, Meclizine HCl, meclofenamicacid, medazepam, medigoxin, medroxyprogesterone acetate, mefenamic acid,Mefloquine HCl, megestrol acetate, melphalan, mepenzolate bromide,meprobamate, meptazinol, mercaptopurine, mesalazine, mesna,mesoridazine, mestranol, methadone, methaqualone, methocarbamol,methoin, methotrexate, methoxsalen, methsuximide, methyclothiazide,methylphenidate, methylphenobarbitone, methyl-p-hydroxybenzoate,methylprednisolone, methyltestosterone, methyprylon, methysergidemaleate, metoclopramide, metolazone, metoprolol, metronidazole,Mianserin HCl, miconazole, midazolam, mifepristone, miglitol,minocycline, minoxidil, mitomycin C, mitotane, mitoxantrone,mofetilmycophenolate, molindone, montelukast, morphine, MoxifloxacinHCl, nabumetone, nadolol, nalbuphine, nalidixic acid, nandrolone,naphthacene, naphthalene, naproxen, naratriptan HCl, natamycin,nelarabine, nelfinavir, nevirapine, nicardipine HCl, nicotin amide,nicotinic acid, nicoumalone, nifedipine, nilutamide, nimodipine,nimorazole, nisoldipine, nitrazepam, nitrofurantoin, nitrofurazone,nizatidine, nofetumomab, norethisterone, norfloxacin, norgestrel,nortriptyline HCl, nystatin, oestradiol, ofloxacin, olanzapine,omeprazole, omoconazole, ondansetron HCl, oprelvekin, ornidazole,oxaliplatin, oxamniquine, oxantelembonate, oxaprozin, oxatomide,oxazepam, oxcarbazepine, oxfendazole, oxiconazole, oxprenolol,oxyphenbutazone, oxyphencyclimine HCl, paclitaxel, palifermin,pamidronate, p-aminosalicylic acid, pantoprazole, paramethadione,paroxetine HCl, pegademase, pegaspargase, pegfilgrastim,pemetrexeddisodium, penicillamine, pentaerythritol tetranitrate,pentazocin, pentazocine, pentobarbital, pentobarbitone, pentostatin,pentoxifylline, perphenazine, perphenazine pimozide, perylene,phenacemide, phenacetin, phenanthrene, phenindione, phenobarbital,phenolbarbitone, phenolphthalein, phenoxybenzamine, phenoxybenzamineHCl, phenoxymethyl penicillin, phensuximide, phenylbutazone, phenytoin,pindolol, pioglitazone, pipobroman, piroxicam, pizotifen maleate,platinum compounds, plicamycin, polyenes, polymyxin B, porfimersodium,posaconazole (Posa), pramipexole, prasterone, pravastatin, praziquantel,prazosin, prazosin HCl, prednisolone, prednisone, primidone,probarbital, probenecid, probucol, procarbazine, prochlorperazine,progesterone, proguanil HCl, promethazine, propofol, propoxur,propranolol, propylparaben, propylthiouracil, prostaglandin,pseudoephedrine, pteridine-2-methyl-thiol, pteridine-2-thiol,pteridine-4-methyl-thiol, pteridine-4-thiol, pteridine-7-methyl-thiol,pteridine-7-thiol, pyrantelembonate, pyrazinamide, pyrene,pyridostigmine, pyrimethamine, quetiapine, quinacrine, quinapril,quinidine, quinidine sulfate, quinine, quininesulfate, rabeprazolesodium, ranitidine HCl, rasburicase, ravuconazole, repaglinide, reposal,reserpine, retinoids, rifabutine, rifampicin, rifapentine, rimexolone,risperidone, ritonavir, rituximab, rizatriptan benzoate, rofecoxib,ropinirole HCl, rosiglitazone, saccharin, salbutamol, salicylamide,salicylic acid, saquinavir, sargramostim, secbutabarbital, secobarbital,sertaconazole, sertindole, sertraline HCl, simvastatin, sirolimus,sorafenib, sparfloxacin, spiramycin, spironolactone, stanolone,stanozolol, stavudine, stilbestrol, streptozocin, strychnine,sulconazole, sulconazole nitrate, sulfacetamide, sulfadiazine,sulfamerazine, sulfamethazine, sulfamethoxazole, sulfanilamide,sulfathiazole, sulindac, sulphabenzamide, sulphacetamide, sulphadiazine,sulphadoxine, sulphafurazole, sulphamerazine, sulpha-methoxazole,sulphapyridine, sulphasalazine, sulphinpyrazone, sulpiride, sulthiame,sumatriptan succinate, sunitinib maleate, tacrine, tacrolimus, talbutal,tamoxifen citrate, tamulosin, targretin, taxanes, tazarotene,telmisartan, temazepam, temozolomide, teniposide, tenoxicam, terazosin,terazosin HCl, terbinafine HCl, terbutaline sulfate, terconazole,terfenadine, testolactone, testosterone, tetracycline,tetrahydrocannabinol, tetroxoprim, thalidomide, thebaine, theobromine,theophylline, thiabendazole, thiamphenicol, thioguanine, thioridazine,thiotepa, thotoin, thymine, tiagabine HCl, tibolone, ticlopidine,tinidazole, tioconazole, tirofiban, tizanidine HCl, tolazamide,tolbutamide, tolcapone, topiramate, topotecan, toremifene, tositumomab,tramadol, trastuzumab, trazodone HCl, tretinoin, triamcinolone,triamterene, triazolam, triazoles, triflupromazine, trimethoprim,trimipramine maleate, triphenylene, troglitazone, tromethamine,tropicamide, trovafloxacin, tybamate, ubidecarenone (coenzyme Q10),undecenoic acid, uracil, uracil mustard, uric acid, valproic acid,valrubicin, valsartan, vancomycin, venlafaxine HCl, vigabatrin,vinbarbital, vinblastine, vincristine, vinorelbine, voriconazole,xanthine, zafirlukast, zidovudine, zileuton, zoledronate, zoledronicacid, zolmitriptan, zolpidem, and zopiclone.

F. KITS

The present disclosure also provides kits. Any of the componentsdisclosed herein may be combined in the form of a kit. In someembodiments, the kits comprise a composition as described above or inthe claims.

The kits will generally include at least one vial, test tube, flask,bottle, syringe or other container, into which a component may beplaced, and preferably, suitably aliquoted. Where there is more than onecomponent in the kit, the kit also will generally contain a second,third or other additional containers into which the additionalcomponents may be separately placed. However, various combinations ofcomponents may be comprised in a container. In some embodiments, all ofthe lipid nanoparticle components are combined in a single container. Inother embodiments, some or all of the lipid nanoparticle components areprovided in separate containers.

The kits of the present disclosure also will typically include packagingfor containing the various containers in close confinement forcommercial sale. Such packaging may include cardboard or injection orblow molded plastic packaging into which the desired containers areretained. A kit may also include instructions for employing the kitcomponents.

Instructions may include variations that can be implemented.

F. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the disclosure. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the disclosure, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe disclosure.

Example 1—Preparation of DOTAP Modified Lipid Nanoparticles

Lipid nanoparticles (LNPs) are the most efficacious carrier class for invivo nucleic acid delivery. Historically, effective LNPs are composed of4 components: an ionizable cationic lipid, zwitterionic phospholipid,cholesterol, and lipid poly(ethylene glycol) (PEG). However, these LNPsresult in only general delivery of nucleic acids, rather than organ ortissue targeted delivery. LNPs typically delivery RNAs only to theliver. Therefore, new formulations of LNPs were sought in an effort toprovide targeted nucleic acid delivery.

The four canonical types of lipids were mixed in a 15:15:30:3 molarratio, with or without the addition of a permanently cationic lipid.Briefly, LNPs were prepared by mixing 5A2-SC8 (ionizable cationic), DOPE(zwitterionic), cholesterol, DMG-PEG, and DOTAP (permanently cationic)in the ratios shown in Table 0.1.

TABLE 0.1 Molar Ratios and Percentages of Lipids in modified LNPS.Lipids/ Molar Ratios Molar Percentage (%) mRNA Name 5A2-SC8 DOPE CholDMG-PEG DOTAP 5A2-SC8 DOPE Chol DMG-PEG DOTAP (wt/wt) mDLNP 15 15 30 3 023.8 23.8 47.6 4.8 0.0 40 DOTAP5 15 15 30 3 3.3 22.6 22.6 45.2 4.5 5.040 DOTAP10 15 15 30 3 7 21.4 21.4 42.9 4.3 10.0 40 DOTAP15 15 15 30 3 1120.3 20.3 40.5 4.1 14.9 40 DOTAP20 15 15 30 3 16 19.0 19.0 38.0 3.8 20.340 DOTAP25 15 15 30 3 21 17.9 17.9 35.7 3.6 25.0 40 DOTAP30 15 15 30 327 16.7 16.7 33.3 3.3 30.0 40 DOTAP35 15 15 30 3 34 15.5 15.5 30.9 3.135.1 40 DOTAP40 15 15 30 3 42 14.3 14.3 28.6 2.9 40.0 40 DOTAP45 15 1530 3 52 13.0 13.0 26.1 2.6 45.2 40 DOTAP50 15 15 30 3 63 11.9 11.9 23.82.4 50.0 40 DOTAP55 15 15 30 3 77 10.7 10.7 21.4 2.1 55.0 40 DOTAP60 1515 30 3 95 9.5 9.5 19.0 1.9 60.1 40 DOTAP65 15 15 30 3 117 8.3 8.3 16.71.7 65.0 40 DOTAP70 15 15 30 3 150 7.0 7.0 14.1 1.4 70.4 40 DOTAP75 1515 30 3 190 5.9 5.9 11.9 1.2 75.1 40 DOTAP80 15 15 30 3 260 4.6 4.6 9.30.9 80.5 40 DOTAP85 15 15 30 3 357 3.6 3.6 7.1 0.7 35.0 40 DOTAP90 15 1530 3 570 2.4 2.4 4.7 0.5 90.0 40 DOTAP95 15 15 30 3 1200 1.2 1.2 2.4 0.295.0 40 DOTAP100 0 0 0 0 100 0.0 0.0 0.0 0.0 100.0 40

For preparation of the mDLNP formulation, 5A2-SC8, DOPE, Cholesterol andDMG-PEG were dissolved in ethanol at the given molar ratios(15:15:30:3). The mRNA was dissolved in citrate buffer (10 mM, pH 4.0).The mRNA was then diluted into the lipids solution to achieve a weightratio of 40:1 (total lipids:mRNA) by rapidly mixing the mRNA into thelipids solution at a volume ratio of 3:1 (mRNA:lipids, v/v). Thissolution was then incubated for 10 min at room temperature. Forformation of DOTAP modified mDLNP formulations, mRNA was dissolved in1×PBS or citrate buffer (10 mM, pH 4.0), and mixed rapidly into ethanolcontaining 5A2-SC8, DOPE, Cholesterol, DMG-PEG and DOTAP, fixing theweight ratio of 40:1 (total lipids:mRNA) and volume ratio of 3:1(mRNA:lipids). As shown in Table 1, each formulation is named DOTAPXwhere X represents the DOTAP molar percentage in total lipids.

Example 2—Characterization of DOTAP Modified mDLNP Formulations

To characterize the different mDLNP formulations, size, polydispersityindex and zeta-potential were examined by dynamic light scattering, 3separate times for each formulation. Size and polydispersity index areshown in FIG. 5A, indicating regardless of DOTAP concentration, theformulations all fell within a size range of about 90 nm to about 160nm, while the polydispersity indices varied from about 0.1 to about 0.3,indicating relative homogeneity of size. Zeta potential is shown foreach formulation in FIG. 5B and shows that the zeta potential generallyincreases with the concentration of DOTAP.

Next, encapsulation efficiency was tested using a Ribogreen RNA assay(Zhao et al., 2016). Briefly, mRNA was encapsulated with about 85%efficiency in mDLNPs without DOTAP (FIG. 5C) when the mRNA was dissolvedin acidic buffer (10 mM citrate, pH 4). Low pH is required to protonatethe ionizable amine in the ionizable cationic lipid (e.g. 5A2-SC8,C12-200, DLin-MC3-DMA) to allow electrostatic complexation withnegatively charged mRNA. For all other formulations on this plot, mixingwas performed at pH 7.4 using mRNA dissolved in PBS. Clearly, with lowconcentrations of DOTAP, encapsulation efficiency was low, but increasedto >80% with a molar percentage of DOTAP above 25% (FIG. 5C). Theencapsulation efficiency was between about 80% and about 95% for allformulations with a molar percentage of greater than 25% DOTAP.Therefore, the potential to use neutral pH PBS mixing is a feature ofthe permanently cationic lipid strategy. This strategy allows for tissuespecific delivery and for high Cas9 protein encapsulation. The additionof a permanently cationic lipid allows formation of LNPs at neutral pH.These encapsulation results are when PBS was used as a buffer. Whenacidic buffers are used (e.g. citrate buffer (10 mM, pH 4.0)), then theencapsulation efficiency is high (>90%) for all formulations from 0 to100% DOTAP.

Finally, pKa was determined using 2-(p-toluidino)-6-naphthalenesulfonicacid (TNS) assay (FIG. 3B) (Zhao et al., 2016). The relationship betweenpKa and tissue specific mRNA delivery was plotted based on the definedrules. Here 8 rules were designed to score as showed in the table.Obviously, liver targeted formulations had a narrow pKa (˜6-7), nosignificant range for spleen targeted formulation, but lung targeteddelivery required high pKa (>9.25). Together distribution and pKadetection into consideration, it was concluded that internal charges ofNPs is one factor that affects mRNA distributions, and thatglobal/apparent LNP pKa is another factor determines the mRNA-mediatedprotein expression profiles in organs

Example 3—Efficacy of Permanently Cationic Lipid Modified mDLNPs formRNA Delivery

To examine the delivery efficacy by which LNPs including a permanentlycationic lipid are able to deliver active cargo in vitro, the DOTAPmodified mDLNPs were loaded with mRNA encoding luciferase, and Huh-7liver cells and A549 adenocarcinomic human alveolar basal epithelialcells were transfected with the 50 ng/well of mRNA. These cells werecultured for 24 hours before luciferase expression and cell viabilitywere examined. As shown in FIG. 6A, DOTAP percentages of 5%-50% werebetter for mRNA delivery and expression in the Huh-7 liver cells invitro, and 10% DOTAP appeared to have the greatest delivery andexpression of luciferase (FIG. 6A). It is noted that deliverycharacteristics in vivo may be different. Generally, these studies maynot be useful to predict in vivo activity, nor tissue tropism due to theadditional in vivo barriers, organ distribution, and cellular specify ofthe SORT LNPs. Additionally, cell viability was examined and it wasfound that 10% DOTAP yielded high viability within the mDLNPs whichexhibited robust luciferase expression (FIG. 6A). Examination of thesame transfection with the A549 lung cancer cell line showed similarresults, with cells transfected with the DOTAP10 formulation exhibitingnearly double the fluorescence of any of the other formulations andmaintaining a high cell viability (FIG. 6A). DOTAP SORT LNPs were formedin PBS (pH 7.4) rather than citric buffer (10 mM, pH 4.0), but mayformed in either buffer system. This is an attribute because it allowsencapsulation and delivery of cargos that are not stable in ethanol oracidic buffer, e.g. proteins.

To determine the effect of ethanol concentration within the formulation,DOTAP25 was selected and prepared with various ethanol to PBS ratios(1:3, 1:5, 1:7.5 ad 1:10) (FIG. 6B). All four formulations showedsimilar encapsulation efficiency, size, and PDI (FIG. 2B). mRNA deliveryefficiency was also measured by transfecting FaDu hypopharynx carcinomacells with 50 ng/well of mRNA in each formulation. The deliveryefficiency of each formulation was similar, with very little effect oncell viability as well (FIG. 6C). Therefore, these formulations appearapplicable to a number of cell types, including liver, lung, and throat.Further, this formulation has also been successfully modified to use1×PBS (pH 7.4) instead of acidic buffer (pH 4.0), and these data showthat the ethanol percentage can be dramatically decreased, which providefor the possibility that DOTAP formulations may deliver cargo that aremuch more sensitive to high ethanol concentrations and acidic buffers,e.g. proteins.

Next, to test the ability of these mDLNPs to deliver mRNA in vivo, micewere injected with a dose of 0.1 mg/kg of Luc mRNA in each of theformulations. FIG. 1B shows ex vivo images of luciferase in major organsat 6 h post IV injection of each formulation. Interestingly, withincreasing molar percentage of DOTAP, luciferase expression moved fromliver to spleen, then to lung, demonstrating organ specific delivery.These data were quantitated, revealing that DOTAP percentage is a factorfor tissue targeting delivery, and that mDLNP (0% DOTAP) is the best forliver delivery, while 5-15% DOTAP are the best for spleen and DOTAP50(50%) is the best for lung delivery (FIG. 1B). Assuming that luciferaseexpression was detected only in liver, spleen and lung after IVinjection, the percentage of luciferase expressed in each organ can becalculated (FIG. 1C). These data clearly indicate that with increasingmolar DOTAP percentage in the formulation, there is decreasing deliveryto and expression in the liver, with close to zero expression seen inthe liver when DOTAP percentage is greater than 70% (FIG. 1B, 1C).However, the greater the DOTAP percentage, the more luminescence is seenin lung tissues, with close to 100% of luminescence seen in the lungwhen DOTAP percentage is greater than 80% (FIG. 1C). Concentrations ofDOTAP 5 and 30 molar percentage showed higher percentages ofluminescence in spleen tissues, while DOTAP10 showed the highestrelative luminescence in the spleen compared to other tissues (FIG. 1C).These results indicate that lipid concentrations can be tailored forspecific tissue delivery, following injection.

To test the organ biodistributions of specific DOTAP formulations ingreater depth, PBS or liver targeting NPs (mDLNP), spleen targeting NPs(DOTAP10) and lung targeting NPs (DOTAP50) were injected into C57BL/6mice (n=2) at a dose of 0.5 mg/kg Cy5-Luc mRNA (dye labeled mRNA totrack RNA LNPs). 6 h post injection, major organs were collected andimaged (FIG. 3A). The organ distribution of formulations was changed bythe amount of DOTAP, and accumulation in liver gradually moved to lungwith increasing DOTAP percentages, but regardless of DOTAP percentage,there are still NPs present in the liver (FIG. 7, FIG. 8). Taking thisdata into account with that of FIG. 1, it is clear that organdistribution is not enough to analyze tissue targeted delivery efficacy(mRNA translation to protein). Further, considering the similar sizedistribution and EE between these formulations, zeta-potential and pKamay play roles in tissue targeting mRNA expression.

In an effort to understand whether the effects of the addition of DOTAPto mDLNPS were limited, or whether the distributions shown above areuniversal to permanent cationic lipid formulated mDLNPs, mDLNPs weregenerated with inclusion of another popular cationic lipid,Didodecyldimethylammonium Bromide (DDAB) (FIG. 2A1). DDAB has twohydrophobic tails with 18 carbons and no unsaturated bonds, and has acompletely different head group than DOTAP (FIG. 1C). DDAB5, DDAB15,DDAB40 and DDAB50 formulations were selected for in vivo delivery (0.1mg/kg, 6 h, n=2). Similar to the DOTAP formulations above, there wasvery little difference in size distribution (FIG. 2A1), even though DDABpercentage in the NPs varied by 10 fold (5% to 50%). Similar to DOTAPNPs, in vivo luciferase expression showed a trend that luminescencemoved from the liver to spleen, then to lung with increasing DDABpercentages (FIG. 2A2).

mDLNPs were formed with a third permanent cationic lipid with theheadgroup 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine chloridewhich has similar structure with DOTAP, but with shorter, 14 carbon,hydrophobic tails ((14:0) EPC) (FIG. 2B1). Similar to the DDAB strategy,(14:0) EPC5, (14:0) EPC15, (14:0) EPC40 and (14:0) EPC50 formulationswere prepared and the size distribution (FIG. 2B1) and in vivo Luc mRNAdelivery (0.1 mg/kg, 6 h, n=2) were examined (FIG. 2B2). Similar to themDLNPs analyzed above, the particle size was generally uniform (FIG.2B1), and as expected, luminescence moved from liver to spleen, then tolung with increasing (14:0) EPC molar percentages (FIG. 2B2). Taking allof these data together, which include different hydrophobic tails,saturated and unsaturated bonds, and different head groups, it appearsthat cationic lipid formulated mDLNP for tissue targeting mRNA deliveryis universal.

In an effort to understand whether the effects of the addition of DOTAPto LNPs were specific to permanently cationic lipids, the effects ofadding zwitterionic lipids instead of permanently cationic lipids inmDLNP formulations was examined. Two representative zwitterionic lipids,phosplolipids with different chemical structures: DSPC and DOCPe weretested. Additionally, it was also tested to determine if the addition ofzwitterionic lipids (instead of permanently cationic lipids) wouldaffect tissue specific delivery efficacy. FIGS. 2C1 and 2D1 show thechemical structures of DSPC and DOCPe lipids (zwitterionic lipids).There are differences in both the positions of the positive and negativecharged functional headgroups and in the hydrophobic domains (saturatedversus unsaturated), suggesting that any observed effect would begeneral/universal for zwitterionic lipids. mDLNPs formulated with eitherDSPC or DOCPe were similar (FIGS. 2C1, 2D1). Interestingly, theinclusion of zwitterionic lipids into 5-component modified DLNPs did notchange the protein expression profile from liver to lungs as was thecase for DOTAP and other permanently cationic lipids. Instead, both DSPCand DOCPe improved mRNA delivery into spleen within given range (lessthan 80% in DSPC and less than 50% in DOCPe). There was no proteinexpression in lungs for any percentages (0.1 mg/kg, 6 h, n=2) (FIGS.2C2, 2D2). Therefore, inclusion of additional zwitterionic lipid can aidspleen delivery, but cannot tune delivery efficacy from liver to spleento lungs like the inclusion of permanently cationic lipids.

In an effort to understand whether the effects of the addition of DOTAPto LNPs were specific to permanently cationic lipids, the effects ofadding ionizable cationic lipids instead of permanently cationic lipidsin mDLNP formulations were examined. Two representative ionizablecationic lipids with different chemical structures: C12-200 and DODAP,were tested. DODAP has a same structure with DOTAP except for the headgroups (quaternary versus tertiary amine). C12-200 is an effectivelipidoid used for siRNA and mRNA delivery containing ionizable tertiaryamines (also no quaternary amines), which has a completely differentstructure with DODAP. (FIGS. 2E1, 2F1) Similarly, the size distributionsof both modified mDLNPs were still uniform at certain percentages (lessthan 80%). (FIGS. 2E1, 2F1) Surprisingly, the inclusion of ionizablecationic lipids into 5-component modified DLNPs did not change theprotein expression profile from liver to spleen to lungs as was the casefor DOTAP and other permanently cationic lipids. Instead, the inclusionof ionizable cationic lipids into DLNPs increased mRNA delivery efficacyof mRNA to the liver. These showed much better delivery efficacy thanoriginal mDLNP (0.1 mg/kg, 6 h, n=2) without additional ionizablecationic lipid (only 5A2-SC8). With increasing percentages of DODAP orC12-200 (50% or 80%), luciferase signal decreased a lot, but liver stillwas the main organ rather than spleen or lung. Therefore, it wasconcluded that organ specific effects can be attributed to inclusionspecific ratios of permanently cationic lipids. Moreover, this datashows that permanently cationic lipids produce different effects thanionizable cationic lipids. These data further show that these trends areuniversal with respect to classes of lipids.

Example 4—CRISPR/Cas9 Gene Editing Using Modified mDLNPs that Co-DeliverCas9 mRNA and sgRNA

First, three sgRNAs targeting Td-Tomato mice were compared to determinewhich sgRNA would be most effective in subsequent experiments. ThesesgRNAs are sgTom1, sgTom2 and sgLoxP. As is shown in FIG. 10A, sgTom1and sgLoxP were delivered and expressed with similar results, and weremore successful at inducing TdTomato than sgTom2 (FIG. 10A). Consideringthe weak PAM of sgLoxP (NAG), sgTom1 was finally chosen for furtherexperiments.

Given the tissue specific mRNA (luc mRNA) delivery shown with DOTAP NPs,and that both DDAB and EPC modified NPs showed similar delivery trends,DOTAP modified mDLNPs were then used for Cas9 mRNA/sgRNA co-deliveryaimed at achieving tissue specific gene editing. To examine co-deliveryin vivo, genetically engineered mice containing a homozygous Rosa26promoter Lox-Stop-Lox tdTomato (tdTO) cassette present in all cells wereused (FIG. 4A). Co-delivery of DOTAP modified mDLNPs housing Cas9-mRNAand sgRNA against LoxP or against Tom enabled deletion of the Stopcassette and induction of tdTO expression (FIG. 4B). The mice were IVinjected by mDLNP and DOTAP50 formulations for co-delivery of IVT Cas9mRNA and modified sgTom1 (4/1, wt/wt) at the total doses of 2.5 mg/kg(50 ug each), then fluorescence of main organs was detected at day 10after treatment. (FIG. 4B). Liver and lung specific CRISPR/Cas geneediting was achieved. Spleen specific editing was also achieved.However, due to very high background red autofluorescence, spleenediting was not quantifiable using this TdTomato reporter mouse.

To further examine tissue specific editing, PTEN was selected as anendogenous target. C57BL/6 mice were IV injected with mDLNP, DODAP20 orDOTAP50 to reach tissue specific gene editing. The total dose was 2.5mg/kg (50 ug each), weight ratio of IVT Cas9 mRNA to modified sgPTEN was4/1, and detection time was day 10 after treatment. sgRNA targeting PTENwas used. A T7E1 assay showed that tissue specific feature was furtherconfirmed with in vivo PTEN editing. (FIG. 4C).

Example 5—CRISPR/Cas9 Gene Editing Using Modified mDLNPs that DeliverCas9 Protein/sgRNA Ribonucleoproteins (RNPs)

Building on the discovery that inclusion of a permanently cationic lipid(e.g. DOTAP) into traditional LNP formulations containing an ionizablecationic lipid, a zwitterionic lipid, cholesterol, and a PEGylatedlipid, it was investigated whether this formulation methodology couldalso deliver other cargoes that are sensitive to ethanol and/or low pHacidic aqueous buffers. The key element of the DOTAP strategy is thatformulations can be prepared using PBS at neutral pH. It was thereforeexamined if this methodology could also encapsulate and deliver largeproteins, such as Cas9, for gene editing applications. DOTNP lipidnanoparticles therefore consist of five components: an ionizablecationic lipids (e.g. 5A2-SC8), a zwitterionic lipid (e.g. DOPE),Cholesterol, DMG-PEG, and modular inclusion of a permanently cationiclipid (e.g. DOTAP). The mole ratio of 5A2-SC8, Cholesterol, DOPE, andDMG-PEG was fixed (15:15:30:5, mol/mol) and DOTNPX means DOTNP withdifferent mole percentage of DOTAP.

The first characterized Cas9/sgRNA complexes were examined if they aresensitive to acidic pH. The size (diameter) (FIG. 11A) and Zetapotential (FIG. 11B) of Cas9/sgLUC complex (mol/mol=1/1) was measured inPBS (pH 7.4) and in citrate Buffer (pH 4.2). The size of Cas9/sgLUCcomplexes prepared in citrate buffer is large (greater than 100 nm) andthe zeta potential is positively charged. These two attributes (sizelarger than a typical efficacious LNP) and positive charge (chargeincompatible to complex with positively charged lipids), render itimpossible to be effectively encapsulated by lipid nanoparticles.However, the size of Cas9/sgLUC complexes prepared in PBS is compact(less than 20 nm) and has negative charges. Therefore, it can beencapsulated by lipid nanoparticles when formulated at neutral pH. Next,Cas9/sgRNA complexes with different Cas9 protein to sgRNA molar ratioswere prepared and characterized. Size (FIG. 11C) and Zeta potential(FIG. 11D) of Cas9/sgLUC complexes prepared with different Cas9/sgRNAmole ratios (1/1, 1/3, and 1/5). Compared with Cas9/sgLUC complex (1/1,mol/mol), higher mole ratios (1/3 and 1/5, mol/mol) showed smaller sizeand more negative charges, which may be beneficial for lipidnanoparticle encapsulation. The compositions were prepared andcharacterized DOTNP lipid nanoparticles after encapsulating Cas9/sgRNAcomplexes. Size (FIG. 11E) and Zeta potential (FIG. 11F) of DOTNP10lipid nanoparticles encapsulating Cas9/sgLUC complex (named DOTNP10-L)when preparing with different mole ratios (1/1, /3, 1/5). This dataindicates encapsulation of Cas9/sgRNA RNPs into monodisperse LNPs. FIG.11G shows TEM images of DOTNP10-L (1/3, mol/mol) LNPs with encapsulatedRNPs. Following this initial study, different sgRNA were used includingsgLUC, sgGFP, sgTOM, and sgPTEN. To distinguish them, the first letterof each gene was added at the end of DOTNP. For example, DOTNP10-L meansDOTNP10 lipid nanoparticles encapsulating Cas9/sgLUC complex; DOTNP10-Gmeans DOTNP10 lipid nanoparticles encapsulating Cas9/sgGFP complex.

TABLE 7 Characterizations of DOTAP10, DSPC50 and DODAP50 formulationsformed by PBS and citric buffer, including size, PDI and encapsulationefficacy. DOTAP10 DSPC50 DODAP50 Citric Citric Citric PBS Buffer PBSBuffer PBS Buffer Size (nm) 155.4 85.1 208.9 202.0 153.5 148.0 PDI 0.090.21 0.30 0.29 0.08 0.15 EE (%) 34.9 54.9 0 43.7 14.4 36.2

TABLE 8 Listed all primers used in this research work, including the lengthof PCR product and their purposes.  Forward Primers  Reverse Primers Name (5′ to 3′) (5′ to 3′) Length Notes Cas9 ATATATGGATCCGCCACCATGGCATATATGAATTCTTA 4233 bp For IVT  CCCAAAGAAGAAGCGGAAGGTC CTTTTTCTTTTTTGCdone CTGGCCGGCCTTTTT CGTGGCCGCCGGCCT TTTGTCGCCTCCCAG Ca9 Seq-1CTGAGCGACATCCTGAGAGTGAAC For  Ca9 Seq-2 AGCAGGTCCTCTCTG sequencing TTCAGto  Ca9 Seq-3 GACGGCTTCGCCAACAGAAACTTC confirm Ca9 Seq-4 TTTGATGCCCTCTTCthe whole GATCCG Cas9  Ca9 Seq-5 GGGAGATCGTGTGGGATAAG sequencesCa9 Seq-6 ACTTCTTAGGGTCCC AGTCC Ca9 Seq-7  AAGAGAGTGATCCTGGCCGAC PTENATCCGTCTTCTCCCCATTCCG GACGAGCTCGCTAAT  638 bp For T7E1 CCAGTG assayIVT sgTom1 TAATACGACTCACTATAGGGAAGT AAAAGCACCGACTCG  120 bp For IVT AAAACCTCTACAAATGGTTTTAGA GTGCC template GCTAGAAATAGC preparation.IVT sgTom2 TAATACGACTCACTATAGGGAAAC AAAAGCACCGACTCG  120 bp Red CTCTACAAATGTGGTAGTTTTAGA GTGCC indicates GCTAGAAATAGC sgRNA IVT sgLoxPTAATACGACTCACTATAGGGCGTA AAAAGCACCGACTCG  120 bp sequencesTAGCATACATTATACGGTTTTAGA GTGCC GCTAGAAATAGC (Cas9 = SEQ ID NOS: 3-4; Ca9Seq-1 = SEQ ID NO: 5; Ca9 Seq-2 = SEQ ID NO: 6; Ca9 Seq-3 = SEQ ID NO:7; Ca9 Seq-4 = SEQ ID NO: 8; Ca9 Seq-5 = SEQ ID NO: 9; Ca9 Seq-6 = SEQID NO: 10; Ca9 Seq-7 = SEQ ID NO: 11; PTEN = SEQ ID NOS: 14-15; IVTsgTom1 = SEQ ID NOS: 44-45; IVT sgTom2 = SEQ ID NOS: 46-47; IVT sgLoxP= SEQ ID NOS: 48-49)

To examine if DOTNP lipid nanoparticles could deliver Cas9/sgRNA RNPcomplexes into the nucleus and mediate efficient gene editing in vitro,a series of experiments were performed. First, DOTNPs containingCas9/sgRNA RNPs that were tagged by a green fluorescent EGFP weretracked by confocal microscopy (FIG. 12A). Images of Hela-Luc cellsafter incubation with DOTNP10 encapsulating Cas9-EGFP/sgLUC complexes(1/3, mol/mol) for 1 h, 3 h, 6 h, and 24 h (9 nM of sgRNA was used)showed that the DOTNPs were internalized into cells and that the Cas9RNP trafficked to the nucleus. Green: EGFP fused Cas9 protein; Blue:nuclei stained with Hoechst 33342. Red arrows indicated the process ofDOTNP10 entering into nucleus. (FIG. 12B)

Next, it was examined if DOTNP lipid nanoparticles could deliverCas9/sgRNA RNP complexes could cut targeted Luciferase DNA. Thepercentages of DNA indels (insertions and deletions) was quantifiedusing the TIDE assay ay the LUC locus following incubation withDOTNP10-L of different mole ratios for 3 days (24 nM of sgRNA was used).DOTNP10 lipid nanoparticles encapsulating Cas9/sgGFP (DOTNP10-G) wasused as negative control. Here, two commercial Cas9 proteins (GeneArtCas9 and Truecut Cas9) were used. (FIG. 12B). Next, a T7EI cleavageassay of Hela-Luc cells incubated with different formulations (24 nM ofsgRNA) was performed to demonstrate DNA editing (FIG. 12C). Among theconditions tested, mole ratio at 1/3 showed the best gene editing whenusing Truecut Cas9 protein. Next, fluorescence microscopy was used totest editing of GFP (FIG. 12D). Images of SKOV3-GFP cells incubated withDOTNP10-L (control, does not target GFP) and DOTNP10-G (does target GFP)(24 nM of sgRNA) which showed on target editing demonstrated bydisappearance of GFP protein expression. Finally, flow cytometry wasused to analyze SKOV3-GFP cells incubated with DOTNP10-L and DOTNP10-G(FIG. 12E). (FIG. 12F) Mean fluorescence intensity of SKOV3-GFP cellsincubated with DOTNP10-L and DOTNP10-G by flow cytometry showed editingof GFP by CRSPR/Cas.

Next, it was examined if DOTNP lipid nanoparticles could deliverCas9/sgRNA RNP complexes in vivo to achieve CRISPR/Cas-mediated geneediting. As before, the genetically engineered TdTomato mouse model wasemployed. 1.5 mg/kg of sgRNA were delivered per mouse with the followingformulations: DOTNP5-T means DOTNP5 LNPs encapsulating Cas9/sgTomcomplex; DOTNP10-T means DOTNP10 LNPs encapsulating Cas9/sgTom complex;DOTNP50-T means DOTNP50 LNPs encapsulating Cas9/sgTom complex. FollowingIV injection of these formulations, tdTomato fluorescence was quantifiedex vivo in major organs 7 days post injection (FIG. 13A). tdTomatofluorescence was only observed in the liver for the DOTNP5-T treatedgroup; In DOTNP10-T group, slight fluorescence was seen in lung and iffurther increasing dose of DOTAP to 50% (DOTNP50-T), most of tdTomatofluorescence was observed in lung. Therefore, similar to the mRNAdelivery experiments summarized above, the DOTAP methodology also allowstissue specific gene editing of Cas9/sgRNA ribonucleoprotein (RNP)complexes. To further examine delivery, LNPs containing sgRNA againstPTEN were delivered. Using the T7EI cleavage assay of liver and lungorgans, it was determined that IV injection of DOTNP5-P (DOTNP5 LNPsencapsulating Cas9/sgPTEN complex), DOTNP10-P (DOTNP10 LNPsencapsulating Cas9/sgPTEN complex) and DOTNP50-P (DOTNP50 LNPsencapsulating Cas9/sgPTEN complex) (2 mg/kg of sgRNA per mouse) mediategene editing (FIG. 13B). The results are consistent with that obtainedby ex vivo imaging. Gene editing was only detected in liver aftertreated with DOTNP5-P; gene editing was obtained both in liver and inlung when incubated with DOTNP10-P; while in DOTNP50-P treatment group,most of gene editing was observed in lung.

The data provided herein show that lipid nanoparticles may be preparedwith varying compositions in order to specifically target the payload tospecific tissues. Particularly, lipid nanoparticles with lowconcentrations of permanently cationic lipids (≤10%) are effective fordelivering nucleic acids to the liver, and LNPs with less than 30%permanently cationic lipid are effective for delivering nucleic acids tothe spleen, LNPs with greater than 30% permanently cationic lipideffectively deliver nucleic acids to the lungs. These findings appear tobe universal, with head groups, saturation, and tail length havinglittle effect.

Example 6—Additional of Another Lipid into Known Four Lipid CompositionsResult in the Change of Delivery Target

The generalizability of the approach (methodology) to include a “fifth”lipid into established 4-component LNPs was then explored.

To examine if the inclusion of a permanently cationic lipid (e.g. DOTAP)could alter the tissue specificity of other ionizable cationic lipids,two well known and well established ionizable cationic lipid LNP systemswere selected. DLin-MC3-DMA was selected and it was formulated withDSPC, Cholesterol, and PEG-DMG. The same molar composition asPatisiran/Onpattro (Alnylam Pharmaceuticals) was produced andsupplemented with 15% or 50% of DOTAP (extra 5th lipid into Onpattro 4lipid formulation) (FIG. 14). DLin-MC3-DMA LNPs are considered the “goldstandard” for both siRNA and mRNA delivery. To date, they have only beenshown to deliver to the liver following IV administration. As shown inFIG. 15A, DOTAP altered mRNA expression profiles in organs forDLin-MC3-DMA based LNPs (0.1 mg/kg Luciferase mRNA, 6 h). Withincreasing percentage of DOTAP, luciferase signal moved from liver tospleen and finally to lung, which was exactly the same with thephenomenon for 5A2-SC8 mDLNPs. To further study the universality of thisapproach, we included DOTAP into C12-200 LNPs (FIG. 16). WhileDLin-MC3-DMA is a two-tailed lipid with a single dimethylamine headgroupthat is considered a stable nucleic acid lipid nanoparticle (SNALP),C12-200 is a representative “lipidoid” that can be formulated intolipid-like LNPs. All three are ionizable cationic lipids. Identical tothe results with 5A2-SC8 and DLin-MC3-DMA, inclusion of 15 or 50% DOTAPinto C12-200 LNPs changed luciferase protein expression following mRNAdelivery from the liver to spleen to lungs (FIG. 15B). Thus, the 5^(th)lipid methodology (such as adding a permanently cationic lipid) isgeneralizable to other ionizable cationic lipid LNPs.

Example 7—Additional of Another Lipid into Known Four Lipid CompositionsResult in Improved Delivery

Furthermore, the generalizability of the approach (methodology) byasking if additional ionizable cationic lipid would improve liverdelivery was explored.

To examine if the ionizable cationic lipid generally promotes liverdelivery, additional 5A2-SC8 ionizable cationic lipids was included asthe “fifth” lipid into LNPs containing appropriate ratios of 5A2-SC8,DOPE, Cholesterol, and PEG DMG. Extra 5A2-SC8 was included atpercentages of 10 to 30 (FIG. 17A and FIG. 18). To avoid saturatedluminescence, a low 0.05 mg/kg dose of mRNA dose was tested (IV, 6 h).As shown in FIG. 17B, both ex vivo images and quantified datademonstrated that increased extra 15% to 25% of 5A2-SC8 did help improvemRNA delivery potency in the liver. 5A2-SC8A20 (5A2-SC8 LNPs plus 20%extra 5A2-SC8) increased Luciferase 2-3 times higher than the originalmDLNP formulation.

Example 8—Studies Relating to Selective Organ Targeting Compositions

This disclosure describes a strategy termed Selective ORgan Targeting(SORT) that allows nanoparticles to be systematically engineered foraccurate delivery of diverse cargoes including mRNA, Cas9 mRNA/sgRNA,and Cas9 ribonucleoprotein (RNP) complexes to the lungs, spleens, andlivers of mice following intravenous (IV) administration (FIG. 19A).Traditional LNPs are composed of ionizable cationic lipids, zwitterionicphospholipids, cholesterol, and poly(ethylene glycol) (PEG) lipids. Thisdisclosure shows that addition of a supplemental component (termed aSORT compound or a selective organ targeting compound) precisely altersthe in vivo RNA delivery profile and mediates tissue specific genedelivery and editing as a function of the percentage and biophysicalproperty of the added SORT lipid. This disclosure shows evidence of atheory for tissue specific delivery, establish that this methodology isuseful for various nanoparticle systems, and provide a method for LNPdesign to edit therapeutically relevant cells.

Effective intracellular delivery materials have conventionally relied onan optimal balance of ionizable amines to bind and release RNAs (pKabetween 6.0-6.5) and nanoparticle stabilizing hydrophobicity (Kanasty etal., 2013; Jayaraman et al., 2012; Nelson et al., 2013; Hao et al.,2015). Without wishing to be bound by any theory, it is believed thatinternal and/or external charge may be a factor for tuning tissuetropism. Intravenous administration of the developed SORT LNPs enabledhigh levels of tissue specific gene editing. SORT is compatible withvarious methods of deploying gene editing machinery, including mRNA,Cas9 mRNA/sgRNA, and Cas9 RNPs (the systemic RNP delivery).Lung-targeted SORT LNPs edited 40% of epithelial cells and 65% ofendothelial cells; spleen-targeted SORT LNPs edited 13% of B cells and10% of T cells; and enhanced liver-targeted SORT LNPs edited 93% ofhepatocytes following a single, low dose injection.

A. Discovery and Development of SORT

To examine the hypothesis that internal charge adjustment could mediatetissue specific delivery, a strategy was conceived to add a 5^(th) lipidto already established LNP compositions with validated efficacy in liverhepatocytes. The rationale was to tune efficacious LNP formulationswithout destroying the core 4-component ratios that are normally usedfor mediating RNA encapsulation and endosomal escape (Wittrup et al.,2015; Cheng et al., 2018).

The effect of adding a permanently cationic lipid (defined as positivelycharged without pKa or pKa>8) to a degradable dendrimer ionizable(pKa<8) cationic lipid named 5A2-SC8 used in mDLNPs (Zhou et al., 2016;Zhang et al., 2018a; Zhang et al., 2018b) was examined, whicheffectively delivered fumarylacetoacetate hydrolase (FAH) mRNA to liverhepatocytes and extended survival in FAH knockout mice (Cheng et al.,2018). This initial base mDLNP formulation consisted of 5A2-SC8,1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol,DMG-PEG (15/15/30/3, mol/mol), and mRNA (5A2-SC8/mRNA, 20/1, wt/wt)(FIG. 20). A series of LNPs were then formed by systematicallyincreasing the percentage of additional permanently cationic lipid from5 to 100% of total lipids (FIGS. 19B & 20).1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), a well-knownquaternary amino lipid, was initially selected as the SORT lipid to addinto LNP formulations. DOTAP-modified SORT formulations thus includedfive lipid components, where 5A2-SC8/DOPE/Chol/DMG-PEG was fixed at15/15/30/3 (mol/mol), and DOTAP was added at molar ratios from 0 to 1200to make a titrated series of formulations (FIG. 20).

The effects of SORT modification was then evaluated by deliveringLuciferase (Luc) mRNA intravenously (IV) at dose of 0.1 mg/kg. Withincreasing molar percentage of DOTAP, resulting luciferase proteinexpression moved progressively from liver to spleen, and then to lung,demonstrating a clear and precise organ specific delivery trend with athreshold that allowed exclusive lung delivery (FIG. 19B). DOTAPpercentage was the key factor that tuned tissue specificity. Base LNPs(0% DOTAP) were optimal for liver delivery, which was anticipated sincethey had been previously optimized for hepatocyte delivery (Cheng etal., 2018). With addition of 10-15% DOTAP, the resulting SORT LNPs couldnow deliver mRNA to cells in the spleen. Increasing the permanentlycationic SORT lipid further, it was found that 50% DOTAP was optimal forlung delivery (FIG. 19C). It is worth noting that although 50% DOTAPSORT LNPs are efficacious for mRNA delivery to the lungs in vivo, theywere not as efficacious for in vitro delivery (FIG. 21). Moreover, 50%DOTAP SORT LNPs possess a neutral zeta potential surface charge (−0.52mV) (FIG. 20), indicating that tissue tropism is not due to positivecharge related MPS uptake. Calculating the relative expression in eachorgan, the use of DOTAP as a SORT lipid completely altered delivery fromliver to lungs (FIG. 19D). Given that it has been estimated that >99% ofcurrent IV nanomedicines are sequestered by the MPS (Wilhelm et al.,2016; Gustafson et al., 2015), these new SORT nanoparticles thereforeovercome one longstanding challenges in nanomedicine.

With the functional role of the permanently cationic SORT lipidelucidated, without wishing to be bound by any theory, it is believedthat inclusion of other lipids may also alter tissue tropism. To explorethis potential, negatively charged 1,2-dioleoyl-sn-glycero-3-phosphate(18PA) was incorporated as a SORT lipid in a similar manner as DOTAP(FIG. 20). At 10-40% 18PA incorporation, SORT LNPs now mediatedcompletely selective delivery to the spleen with no luciferaseexpression in any other organ (FIG. 19E). Thus, negatively charged SORTlipids allow for explicit delivery to the spleen. These resultsindicated that SORT lipid percentage can be tailored for specific tissuemRNA delivery via IV injection.

B. SORT is Generalizable to Other LNP Types and Lipid Classes

It was then explored if the SORT methodology could be applied to otherclasses of established 4-component LNPs to test whether SORT isuniversal. First, DLin-MC3-DMA was formulated with DSPC, cholesterol,and PEG-DMG with the same molar composition as FDA-approved Onpattro(Patisiran) (30) (FIG. 22), considered the “gold standard” for bothsiRNA and mRNA delivery. To date, they have only been shown to deliverto the liver following IV administration, which was confirmed here aswell (FIG. 19F). As expected, supplementing DLin-MC3-DMA LNPs with DOTAPaltered the protein expression profile of the Onpattro formulation. Withincreasing SORT lipid percentage, the luciferase signal moved from theliver to spleen to lung, which was exactly the same phenomenon for theinitially tested 5A2-SC8 DLNPs. To further study the universality of theapproach, DOTAP was included into C12-200 LNPs (FIGS. 19G & 22), whichare also well validated for RNA delivery to the liver (Kove et al.,2010; Kauffman et al., 2015). Identical to the results with 5A2-SC8 andDLin-MC3-DMA LNPs, inclusion of 15 or 50% DOTAP into C12-200 LNPschanged luciferase protein expression following mRNA delivery from theliver to spleen to lungs (FIGS. 19G & 22). Additionally, inclusion of18PA as a SORT lipid mirrored the results with 5A2-SC8 and mediatedexclusive delivery of Luc mRNA to the spleen for both DLin-MC3-DMASNALPs and C12-200 LLNPs (FIGS. 19F-G). While DLin-MC3-DMA is atwo-tailed lipid with a single dimethylamine headgroup that forms stablenucleic acid lipid nanoparticles (SNALPs), C12-200 is a representativelipidoid that forms lipid-like LNPs (LLNPs). Thus, the SORT methodologywas shown to be generalizable to other classes of ionizable cationiclipid LNPs, which will allow existing liver-targeting LNPs to be easilyaltered to deliver mRNA to the spleen or lungs. Specifically, the SORTtechnology may allow FDA-approved Onpattro to be quickly redeveloped fortreatment of diseases in the lung and spleen.

To understand whether the tissue tropism profiles observed were specificto exact chemical structures or generalizable to defined chemicalclasses, multiple permanently cationic, anionic, zwitterionic, andionizable cationic SORT lipids were evaluated (FIG. 23). First, 5A2-SC8LNPs was generated with two additional permanently cationic lipids:didodecyldimethylammonium bromide (DDAB) and1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine chloride (EPC). Theselipids all contain quaternary amino groups, but there are major chemicaldifferences in the polar head group, linker region, and hydrophobicdomain (e.g. degree of saturation). LNPs containing 5, 15, 40, 50, and100% DDAB or EPC were formulated and characterized. The in vivoluciferase expression profile matched that of the DOTAP LNPs, where theluminescence activity systematically shifted from the liver to spleen,then to lung with increasing DDAB or EPC percentages (0.1 mg/kg, 6 h).Once the percentage was increased to 40%, high luciferase signal wasobserved exclusively in the lungs (FIG. 23A).1,2-dimyristoyl-sn-glycero-3-phosphate (14PA) andsn-(3-oleoyl-2-hydroxy)-glycerol-1-phospho-sn-3′-(1′,2′-dioleoyl)-glycerol(18BMP) was generated as representative anionic lipids with verydifferent structures compared to 18PA. All anionic SORT lipids promotedexclusive delivery to the spleen (FIG. 23B). This flexibility providesways to balance multiple factors including potency, selectivity, andtolerability by optimizing the SORT compound.

Inspired by these findings, other ionizable cationic lipids were addedto established formulations. As expected, addition of DODAP or C12-200to 5A2-SC8 LNPs did not significantly alter tissue tropism, butsurprisingly did enhance liver delivery >10-fold at 20% incorporation(FIG. 23C). Supplementing already established 5A2-SC8 LNPs withadditional 5A2-SC8 as a SORT lipid dramatically improved liver mRNAdelivery, producing 10⁷ photons/sec/cm² at the extremely low dose of0.05 mg/kg. SORT thus offers a new strategy to further improve livertargeting LNP systems (FIG. 24). The effect of using zwitterionic lipids(DOCPe and DSPC) were also evaluated as SORT lipids. While the tissuetropism was found to move from liver to spleen, it was not as selectivecompared to use of cationic or anionic SORT lipids (FIG. 25).

To test the limits of the SORT methodology, it was examined if SORTcould “activate” otherwise inactive formulations. Indeed, supplementinga completely inactive formulation with DODAP or DOTAP resulted in tissuespecific delivery to the spleen and lung (FIG. 26). Taking these resultstogether, the SORT is a modular and universal strategy to achieve tissuetargeted delivery.

C. SORT Alters Protein Corona, LNP Biodistribution, and Apparent pKa toMediate Organ Specific Delivery

Mechanistic experiments were conducted to explore how and why inclusionof extra lipids in defined categories controls mRNA delivery todifferent organs. It is logical that LNPs that deliver to cells withinthe lungs should biodistribute (accumulate) in the lungs. Cy5-labeledmRNA was delivered to track the in vivo distribution of 5A2-SC8 LNPscontaining SORT lipids with lung (DOTAP quaternary amino lipid), spleen(18PA anionic lipid and DSPC zwitterionic lipid) and liver (DODAPionizable tertiary amino lipid) tropism (FIGS. 27A & 28). All LNPs wereinjected IV at a dose of 0.5 mg/kg Cy5-labeled mRNA and imaging wasperformed after 6 hours. As shown in FIG. 27A, DOTAP alteredbiodistribution with a progressive increase in lung accumulation as afunction of DOTAP percentage. Incorporation of 18PA increased uptakeinto the spleen. DODAP slightly increased liver and decreased spleenaccumulation. Interestingly, there was no protein expression at all inthe liver for lung- and spleen-specific SORT LNPs, even though theseLNPs still accumulated in the liver. This suggests that organbiodistribution is required for organ specific efficacy but is not theonly factor to explain the mechanism of tissue targeted delivery.

Without wishing to be bound by any theory, it is believed that thechanges in biodistribution and activity in defined cell populationscould be due to alteration of the protein corona, where the binding ofspecific protein(s) creates a functionally active biological identity.Using quantitative mass spectroscopy analysis, it was found that theaddition of SORT molecules dramatically changes both the specificproteins that most tightly bind and the overall protein coronacomposition. Without wishing to be bound by any theory it is believedthat lung-specific SORT LNPs selectively and most abundantly boundvitronectin, which can interact with positively-charged lipids and bindsαvβ integrins that are highly expressed on both endothelial andepithelial cells of the lungs. This mechanism of endogenous targetingdraws a direct comparison to adenoviruses that exploit the αvβ5 integrinto target the bronchial epithelium. Spleen-specific SORT LNPs mosttightly bound β2-glycoprotein I, which has been shown to interact withnegatively-charged lipids and potentially plays a role in spleenlocalization with immune cell populations in the spleen. It is worthnoting that the complex mixture of bound proteins may also play a role.This ensemble effect was also viewed as a potential mechanism fortargeting of multiple cell types and as a way to further enrichspecificity to specific cell types within one organ using alternativeSORT molecules. It has previously been shown that Apolipoprotein E bindsto DLin-MC3-DMA Onpattro LNPs and that efficacy is lost in Apo Eknockout animals. Thus, there is strong evidence that Apo E is requiredfor receptor-mediated targeting and uptake in hepatocytes, presumably byLDL receptor, and that the described protein corona mechanism cancontrol cellular specificity and efficacy. To see that mDLNPs alsostrongly associate with Apo E was therefore encouraging, providingfurther evidence of their hepatocyte efficacy and strengthening thevalidity of the protein corona assays. Liver-enhanced SORT LNPs retainApo E binding but also are enriched in albumin, suggesting a possibleexpansion of cell types within the liver. These data cumulatively showthat the chemical structure of the SORT molecule can direct a specificprotein corona that alters organ tropism and cellular specificity.Without wishing to be bound by any theory, it is believed that theidentity of the SORT molecule can control the protein corona identitysuggesting that SORT molecules could include a sugar, a lipid, a smallmolecule therapeutic agent, a vitamin, small molecules, hydrophilicmolecules, hydrophobic molecules, amphipathic molecules, peptides,protein, and the like.

The apparent/global pK_(a) was examined because it has been establishedas a parameter that correlates LNPs with functional activity. Forexample, it has been shown that pK_(a) around 6.4 is optimal fordelivery to hepatocytes (Jayaraman et al., 2012). The apparent pK_(a)was analyzed using the TNS assay for all efficacious in vivoformulations (67 LNPs) (FIGS. 27B & 29, Table 1). Because SORT involvesinclusion of additional charged lipids, the resulting TNS titrationcurves capture the ionization behavior of more complex mixed speciesLNPs. As such, the relative pK_(a) was instead estimated when 50% ofnormalized signal was produced. When plotting relative pK_(a) withrespect to tissue tropism, SORT LNPs grouped into defined apparentpK_(a) ranges (FIG. 27B). As expected, all efficacious liver targetedformulations had a very narrow pK_(a) within the well-established 6-7range (Jayaraman et al., 2012). All lung targeted formulations werelocated in the high pK_(a) range (>9). Conversely, spleen tropic SORTLNPs grouped to the low pK_(a) range (2-6). These results confirm that6-7 is optimal for delivery to the liver but reveal the finding thathigh pK_(a) mediates lung delivery and low pK_(a) mediates spleendelivery. It should be note that all SORT LNPs still contain ionizablecationic lipids, which are considered useful for endosomal escape(Wittrup et al., 2015) due to their ability to acquire charge. Controlexperiments were performed and confirmed that inclusion of an ionizablecationic lipid was required for efficacy (FIG. 30). Thus, SORT allowsretention of the molecules with specific microspecies pK_(a) that arerequired for efficacy in desired molar proportions, while inclusion ofSORT lipids modifies the apparent pK_(a). Without wishing to be bound byany theory, it is believed a two-part mechanism may play a role. SORTLNPs selectively bind specific proteins in the serum that enablereceptor-mediated efficacy in cells in the lungs or spleen, very similarto how lipoprotein particles (e.g. LDL) natively transport cholesterol.This controlled and predictable endogenous targeting mechanism enablesSORT LNPs to reach non-liver targets. The second part involves how theSORT molecules alter the properties of the non-hepatic targeting SORTLNPs such that they no longer possess the physiochemical properties forliver efficacy (e.g. global/apparent pK_(a) 6.4), which provides theprecision. It is also noted that other and more complicated factors,such as cell-specific endocytic trafficking differences, may also play arole. Taking results into consideration, it is suggested that theinternal charges of LNP nanostructures mediate biodistribution and thatthe apparent pK_(a) correlates with protein expression profiles inspecific organs. This particular value may be used to continue todevelop other organ-specific nanoparticles.

TABLE 1 Details of DDAB, EPC, 14PA, 18BMP, DODAP, C12-200, 5A2-SC8,DSPC, and DOCPe modified mDLNP formulations (SORT LNPs), including molarratio and percentage of each component, weight ratios of total lipids tomRNA, size, and PDI. Molar Ratios Molar Percentage (%) Lipids/ 5A2- DMG-5A2- DMG- mRNA Size Name SC8 DOPE Chol PEG X^(a) SC8 DOPE Chol PEG X^(a)(wt/wt) (nm) PDI  5% DDAB 15 15 30 3 3.315 22.62 22.62 45.24 4.52 5.0040 193.5 0.14  15% DDAB 15 15 30 3 11.12 20.24 20.24 40.47 4.05 15.00 40167.7 0.13  40% DDAB 15 15 30 3 42 14.29 14.29 28.57 2.86 40.00 40 143.80.13  50% DDAB 15 15 30 3 63 11.90 11.90 23.81 2.38 50.00 40 174.7 0.21100% DDAB 0 0 0 0 100 0.00 0.00 0.00 0.00 100.00 40 3174.3 0.23  5% EPC15 15 30 3 3.315 22.62 22.62 45.24 4.52 5.00 40 195.3 0.14  15% EPC 1515 30 3 11.12 20.24 20.24 40.47 4.05 15.00 40 166.3 0.14  40% EPC 15 1530 3 42 14.29 14.29 28.57 2.86 40.00 40 116.7 0.15  50% EPC 15 15 30 363 11.90 11.90 23.81 2.38 50.00 40 105.7 0.17 100% EPC 0 0 0 0 100 0.000.00 0.00 0.00 100.00 40 455.9 0.32  5% 14PA 15 15 30 3 3.316 22.6222.62 45.24 4.52 5.00 40 92.8 0.18  10% 14PA 15 15 30 3 7 21.43 21.4342.86 4.29 10.00 40 98.6 0.18  20% 14PA 15 15 30 3 15.75 19.05 19.0538.10 3.81 20.00 40 105.7 0.17  30% 14PA 15 15 30 3 27 16.67 16.67 33.333.33 30.00 40 107.3 0.19 100% 14PA 0 0 0 0 100 0.00 0.00 0.00 0.00100.00 40 2607.3 0.28  5% 18BMP 15 15 30 3 3.315 22.62 22.62 45.24 4.525.00 40 112.0 0.13  10% 18BMP 15 15 30 3 7 21.43 21.43 42.86 4.29 10.0040 131.4 0.10  20% 18BMP 15 15 30 3 15.75 19.05 19.05 38.10 3.81 20.0040 172.9 0.12  30% 18BMP 15 15 30 3 27 16.67 16.67 33.33 3.33 30.00 40195.9 0.13 100% 18BMP 0 0 0 0 100 0.00 0.00 0.00 0.00 100.00 40 87.40.21  10% DODAP 15 15 30 3 7 21.43 21.43 42.86 4.29 10.00 40 138.5 0.11 20% DODAP 15 15 30 3 15.75 19.05 19.05 38.10 3.81 20.00 40 122.4 0.18 50% DODAP 15 15 30 3 63 11.90 11.90 23.81 2.38 50 00 40 148.0 0.15  80%DODAP 15 15 30 3 252 4.76 4.76 9.52 0.95 80.00 40 180.4 0.13 100% DODAP0 0 0 0 100 0.00 0.00 9.00 0.00 100.00 40 932.8 0.74  10% C12-200 15 1530 3 7 21.43 21.43 42.86 4.29 10.00 40 179.7 0.11  20% C12-200 15 15 303 15.75 19.05 19.05 38.10 3.81 20.00 40 141.3 0.19  50% C12-200 15 15 303 63 11.90 11.90 23.81 2.38 50.00 40 156.1 0.16  80% C12-200 15 15 30 3252 4.76 4.76 9.52 0.95 80.00 40 273.2 0.21 100% C12-200 0 0 0 0 1000.00 0.00 0.00 0.00 100.00 40 2505.3 1.00 10% 5A2-SC8 15 15 30 3 7 21.4321.43 42.86 4.29 10.00 40 127.1 0.14 15% 5A2-SCS 15 15 30 3 11.12 20.2420.24 40.47 4.05 15.00 40 130.9 0.13 20% 5A2-SC8 15 15 30 3 15.75 19.0519.05 38.10 3.81 20.00 40 137.6 0.10 25% 5A2-SC8 15 15 30 3 21 17.8617.86 35.71 3.57 25.00 40 126.0 0.12 30% 5A2-SC8 15 15 30 3 27 16.6716.67 33.33 3.33 30.00 40 134.7 0.10  20% DSPC 15 15 30 3 15.75 19.0519.05 38.10 3.81 20.00 40 115.7 0.19  50% DSPC 15 15 30 3 63 11.90 11.9023.81 2.38 50.00 40 202.0 0.29  80% DSPC 15 15 30 3 252 4.76 4.76 9.520.95 80.00 40 1024.7 0.67 100% DSPC 0 0 0 0 100 0.00 0.00 0.00 0.00100.00 40 2134.8 0.99  20% DOCPe 15 15 30 3 15.75 19.05 19.05 38.10 3.8120.00 40 147.7 0.15  50% DOCPe 15 15 30 3 63 11.90 11.90 23.81 2.3850.00 40 173.6 0.20  80% DOCPe 15 15 30 3 252 4.76 4.76 9.52 0.95 80.0040 127.7 0.21 100% DOCPe 0 0 0 0 100 0.00 0.00 0.00 0.00 100.00 40 93.70.36 ^(a)X represents DDAB, EPC, 14PA, 18BMP, DODAP, C12-200, 5A2-SC8,DSPC and DOCPe.

D. SORT Allows Lung, Liver, and Spleen Specific Gene Editing FollowingIV Administration

Given the ability of SORT LNPs to target specific organs, these findingwere then applied to tissue specific gene editing via IV injection. TheCRISPR/Cas (clustered regularly interspaced short palindromicrepeat/CRISPR-associated protein (Cas)) technology can edit the genomein a precise and sequence dependent manner and has rapidly developed foruse in diverse applications, including for potential correction ofdisease-causing mutations (Jinek et al., 2012; Cong et al., 2013; Maliet al., 2013; Hendel et al., 2015; Yin et al., 2016; Yin et al., 2017;Wang et al., 2018; Amoasii et al., 2018). Gene editing can be achievedby local administration injections (Zuris et al., 2015; Sun et al.,2015; Chew et al., 2016; Staahl et al., 2017). However, many seriousgenetic disorders arise from mutations in cells deep in organs, wherecorrection of specific cells will be required to cure disease. Suchcorrection may be best achieved by systemic administration. It has beenrecently reported that IV co-delivery of Cas9 mRNA and sgRNA is a safeand effective strategy to enable gene editing (Miller et al., 2017; Yinet al., 2017; Finn et al., 2018). To date, however, there have been noreports of LNPs rationally engineered to edit cells in organs outside ofthe liver.

To examine and quantify the ability of SORT LNPs to mediateorgan-specific gene editing, genetically engineered tdTomato (tdTom)reporter mice containing a LoxP flanked stop cassette (Tabebordbar etal., 2016) were utilized that prevents expression of the tdTom protein(Staahl et al., 2017). Once the stop cassette is deleted, tdTomfluorescence is turned on, allowing detection of gene edited cells (FIG.31A). Cre recombinase mRNA (Cre mRNA) were initially delivered toactivate tdTom in edited cells. Fluorescent tissues were readilyapparent (FIG. 31B) in selected organs treated with liver, lung, andspleen selective SORT LNPs. It should be noted that separate controlshad to be used for each experiment because these mice have somebackground organ fluorecense, which is weakest in the spleen compared toother organs (FIGS. 31C & 32). This makes detection of spleenspecificity more challenging to differentiate in the tdTom mouse model.When endogenous PTEN was subsequently edited, spleen specific SORT LNPsshowed clean DNA cutting by the T7E1 assay only in the spleen (FIG. 33C)without any cutting of DNA in the liver or lungs. Nevertheless, tdTompositive cells were easily seen by confocal imaging of tissue sections(FIG. 31D).

E. SORT Enables High Levels of Editing in Specific and TherapeuticallyRelevant Cell Populations

Gene editing of specific cell types within the liver, lung, and spleenusing flow cytometry of single cells extracted from edited organs werequantified (FIG. 31E). Liver-specific SORT (20% DODAP) 5A2-SC8 LNPsedited ˜93% of all hepatocytes in the liver following a single injectionof 0.3 mg/kg Cre mRNA (FIGS. 31E & 34). This is the highest level ofhepatocyte gene editing reported to date. Lung-specific SORT (50% DOTAP)5A2-SC8 LNPs edited ˜40% of all epithelial cells, ˜65% of allendothelial cells, and ˜20% of immune cells in the lungs at the samedose (FIGS. 31E & 35). Given that epithelial cells are a primary targetfor correction of mutations in CFTR that cause cystic fibrosis, thisresult establishes lung-specific SORT LNPs as a compelling deliverysystem with immediate application for correcting CFTR mutations.Finally, spleen-specific SORT (30% 18PA) 5A2-SC8 LNPs edited ˜13% of allB cells, ˜10% of all T cells, and ˜20% of all macrophages (FIGS. 31E &36). Due to the improved selectivity over prior studies, spleen-specificSORT LNPs could be applicable to treat non-Hodgkin's B cell lymphoma andother immune disorders. Although the initial focus was on single, lowdose injection quantification, higher levels of editing are achievableby administering higher doses or multiple injections.

F. SORT Allows Tissue Specific Gene Editing Via IV Co-Delivery of Cas9mRNA/sgRNA and by Delivery of Cas9 RNPs

The ability of SORT LNPs to achieve tissue specific CRISPR/Cas geneediting via IV co-delivery of Cas9 mRNA and sgRNA in a singlenanoparticle (FIGS. 37A, 38, & 39, Table 2) were next examined. Theliver and lung targeting SORT LNPs were injected at a dose of 2.5 mg/kgtotal RNA (4:1 mRNA:sgRNA, wt:wt) and quantified gene editing 10 daysfollowing a single IV injection. As shown in FIG. 37B, strong tdTomfluorescence in the liver was observed for both the base LNP and 20%DODAP SORT LNP treated mice, and strong fluorescence in the lung of 50%DOTAP SORT LNP treated mice. All results were consistent with the LucmRNA delivery results. Due to fast turnover of splenic immune cells inmice (Kamath et al., 2000), the weight ratio of Cas9/sgRNA was optimizedto be 2/1 (FIG. 39) and tested the spleen editing two days afterinjection. Accounting for background autofluorescence, bright tdTomfluorescence was observed in the spleen of 30% 18PA-treated mice, andclear T7E1 cleavage bands were detected exclusively in DNA isolated fromthe spleen (no editing of liver or lungs) (FIG. 33). The fluorescencewas then confirmed by imaging tissue sections with confocal microscopy(FIG. 37C).

Next, the direct delivery of Cas9 RNPs was explored, which is the mostchallenging strategy for synthetic carriers. The use of permanentlycationic SORT lipids enabled Cas9 protein/sgtdTom complexes to beencapsulated with control over tissue tropism. IV injection of 7% DOTAPSORT LNPs enabled liver editing, while 55% DOTAP SORT LNPs enabledexclusive lung editing (FIG. 37F). These data indicate that thedescribed methodology enables liver, lung, and spleen specificCRISPR/Cas gene editing.

To go beyond reporter mice, the ability of tissue specific LNPs to editan endogenous target was tested. PTEN was selected because it is awell-established tumor suppressor expressed in most cells. Wild typeC57BL/6 mice were injected with SORT LNPs co-loaded with Cas9 mRNA andsgPTEN (2.5 mg/kg total RNA). Generation of insertions and deletions(indels) was quantified 10 days following a single IV injection. Asshown in FIG. 37D, clear DNA cleavage bands were observed in specifictissues by T7E1 assay which demonstrated that both base LNPs and 20%DODAP SORT LNPs mediated effective PTEN editing in liver, but not at allin lung or spleen. Remarkably, 50% DOTAP SORT LNPs showed PTEN editingexclusively in the lungs. To further confirm PTEN editing, H&E stainingand immunohistochemistry (IHC) of tissue sections was performed. Asshown in FIG. 37E, cells in tissue sections obviously displayed clearcytoplasm, which is a known phenotype of PTEN loss due to lipidaccumulation (Xue et al., 2014). Moreover, negative staining of PTEN wasobserved in IHC sections in both liver and lung tissues, providing clearevidence for PTEN editing. Although spleen specific 18PA SORT LNPediting was more challenging to distinguish in the tdTom mouse model,clear spleen PTEN editing could be observed in wild type mice with theoptimized weight ratio of Cas9/sgPTEN (2/1) and detection time (2 days).No editing of DNA in the liver or lungs was observed by the T7E1 assayperformed on 18PA SORT LNP injected mice (FIG. 33). Finally, SORT wasapplied to Cas9 RNPs and examined endogenous editing of PTEN. As before,7% and 55% DOTAP SORT LNPs containing Cas9 protein/sgPTEN enabled liverand lung specific editing, respectively (FIG. 37G). These results,targeting an endogenous gene, demonstrate rationally guided tissueselective gene editing achieved by synthetic carriers.

G. Materials and Methods

I. Materials

5A2-SC8 (Zhou et al., 2016), DLin-MC3-DMA (Jayaraman et al., 2012), andC12-200 (Love et al., 2010) were synthesized and purified by followingpublished protocols. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),dimethyldioctadecylammonium (DDAB),1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC),1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt) (18PA),1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt) (14PA),sn-(3-oleoyl-2-hydroxy)-glycerol-1-phospho-sn-3′-(1′,2′-dioleoyl)-glycerol(ammonium salt) (18:1 Hemi BMP, 18BMP),1,2-dioleoyl-3-dimethylammonium-propane (DODAP),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethyl phosphate(DOCPe) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) werepurchased from Avanti Polar Lipids. Cholesterol was purchased fromSigma-Aldrich. 1,2-Dimyristoyl-sn-glycerol-methoxy(poly((ethyleneglycol) MW 2000) (DMG-PEG2000) was purchased from NOF AmericaCorporation. Cas9 protein was purchased from Thermo Fisher. TheONE-Glo+Tox Luciferase Reporter assay kit was purchased from PromegaCorporation. Pur-A-Lyzer Midi Dialysis Kits (WMCO, 3.5 kDa) werepurchased from Sigma-Aldrich. 4′,6-Diamidino-2-phenylindoledihydrochloride (DAPI) was purchased from Thermo Fisher Scientific. Cas9mRNA was produced by in vitro translation (CVT). Cy5-labelled fireflyluciferase mRNA (Cy5-Luc mRNA), unlabeled firefly luciferase mRNA (LucmRNA), and mCherry mRNA were purchased from TbLink BioTechnologies.D-Luciferin (Sodium Salt) was purchased from Gold Biotechnology.Modified sgTom1 and sgPTEN (Table 2) were purchased from Synthego.

TABLE 2 Relative apparent pK_(a) values of SORT LNPs measured by the TNSassay. MC3 and C12-200 mDLNP based LNPs based LNPs C1 based LNPs NamePKa Name pKa Name pKa Name pKa Name pKa Name pKa mDLMP   6.46 15%   7.2110% 5.15 50% 6.89 MC3   6.28 20%   5.61 O O DDAB C 14PA A C12- I LNPs ODODAP I C A 200 O C1 I I  5%   6.67 40% >11.0 20% 4.78 10% 6.34 15%  6.86 30%   5.53 DOTAP C DDAB C 14PA A 5A2- I DOT C DODAP I C C A SC8MC3 C1 I C I 10%   6.92 50% >11.0 30% 3.73 15% 6.40 50%   >11 10%   5.89DOTAP C DDAB C 14PA A 5A2- I DOT C DOTAP C C C A SC8 MC3 C1 I C C 15%  7.48  5%   6.51  5% 5.22 20% 6.44 10%   6.12 20%   7.55 DOTAP C EPC C18BMP A 5A2- I 18PA A DOTAP C C C A SC8 MC3 C1 I A C 20%   8.49 15%  6.76 10% 4.93 25% 6.49 20%   5.66 30%   >11 DOTAP C EPC C 18BMP A 5A2-I 18PA A DOTAP C C C A SC8 MC3 C1 I A C 30%   10.6 40%   9.45 20% 4.6330% 6.48 30%   5.21 DOTAP C EPC C 18BMP A 5A2- I 18PA A C C A SC8 MC3 IA 40% >11.0 50% >11.0 30% 4.09 20% 6.40 C12-   6.64 DOTAP C EPC C 18BMPA DSPC Z 200 O C C A Z LNPs O 50% >11.0  5%   4.53 10% 6.22 50% 6.09 15%  6.77 DOTAP C 18PA A DODAP I DSPC Z DOT C C A I Z C12- 200 C 60% >11.010%   4.44 20% 6.46 80% 4.84 50%   >11 DOTAP C 18PA A DODAP I DSPC Z DOTC C A I Z C12- 200 C 70% >11.0 20%   4.43 50% 6.44 20% 6.78 10%   5.95DOTAP C 18PA A DODAP I DOCPe Z 18PA A C A I Z C12- 200 A 80% >11.0 30%  3.97 80% 6.09 50% 9.05 20%   5.69 DOTAP C 18PA A DODAP I DOCPe Z 18PAA C A I Z C12- 200 A 90% >11.0 40%   3.52 10% 6.58 30%   5.55 DOTAP C18PA A C12-200 I 18PA A C A I C12- 200 A  5%   6.55  5%   5.34 20% 6.96DDAB C 14PA A C12-200 I C A I O = Original LNPs C = Cationic Lipids +LNPs A = Anionic Lipids + LNPs I = Ionizable Lipids + LNPs Z =Zwitterionic Lipids + LNPs

II. Nanoparticle Formation

RNA-loaded LNP formulations were formed using the ethanol dilutionmethod (Zhou et al., 2016). The liver-targeted mRNA formulation (mDLNP)was developed and reported in a previous paper (Cheng et al., 2018), andthe base formulations were prepared as previously described (Jayaramanet al., 2012; Love et al., 2010). Unless otherwise stated, all lipidswith specified molar ratios were dissolved in ethanol and RNA wasdissolved in 10 mM citrate buffer (pH 4.0). The two solutions wererapidly mixed at an aqueous to ethanol ration of 3:1 by volume (3:1,aq.:ethanol, vol:vol) to satisfy a final weight ratio of 40:1 (totallipids:mRNA), then incubated for 10 min at room temperature. To prepareSORT LNP formulations containing anionic SORT lipids (such as 18PA, 14PAand 18BMP), the anionic lipids were dissolved in tetrahydrofuran (THF)first then mixed with other lipid components in ethanol, finallyyielding formulations with mRNA buffer (10 mM, pH 3.0) as describedabove. All formulations were named based on the additional lipids.Taking DOTAP mDLNP as an example, the internal molar ratio of mDLNP wasfixed as reported in a published paper with5A2-SC8/DOPE/Cholesterol/DMG-PEG of 15/15/30/3 (Cheng et al., 2018).DOTAP, as the additional lipid, was dissolved into the above ethanollipid mixture with specified amount, making the molar ratio of5A2-SC8/DOPE/Cholesterol/DMG-PEG/DOTAP equal to 15/15/30/3/X, thenrapidly mixed with aq. mRNA solutions following the above standardprotocol, finally producing SORT LNPs named Y % DOTAP, where Y means themolar percent of DOTAP in total lipids. Formulations with otheradditional lipids were formed similarly with the above methods (FIG. 20and Table 3). For Cas9/sgRNA ribonucleoprotein (RNP) encapsulation,1×PBS was used for formulation and the molar ratio of Cas9 and sgRNA wasfixed at 1:3. After SORT LNP formation, the fresh LNP formulations werediluted with 1×PBS to 0.5 ng/μL mRNA (with final ethanol concentration<5%) for in vitro assays and size detection. For in vivo experiments,the formulations were dialyzed (Pur-A-Lyzer Midi Dialysis Kits, WMCO 3.5kDa, Sigma-Aldrich) against 1×PBS for 2 h and diluted with PBS to 15μL/g for intravenous (IV) injections.

TABLE 3  sgRNA sequences Sequences  PAM Name (5′ to 3′) (5′ to 3′) NotessgTom1 AAGTAAAACCTCTACAAA TGG Chemical  TG (SEQ ID NO: 1)modified sgTom1 sgPTEN AGATCGTTAGCAGAAACA AGG and sgPTENAA (SEQ ID NO: 2) were selected

III. Characterization of mRNA Formulations

Size distribution and polydispersity index (PDI) were measured usingDynamic Light Scattering (DLS, Malvern MicroV model; He—Ne laser, λ=632nm), zeta-potential was measured after diluting with 1×PBS. To measurethe apparent pK_(a) of mRNA formulations, the2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) assay was employed(Cheng et al., 2018; McLaughlin and Harary, 1976; Bailey and Cullis,1994; Heyes et al., 2005), with some modification. mRNA formulations (60μM total lipids) and the TNS probe (2 μM) were incubated for 5 min witha series of buffers, containing 10 mM HEPES, 10 mM MES(4-morpholineethanesulfonic acid), 10 mM ammonium acetate and 130 mMNaCl (the pH ranged from 2.5 to 11). The mean fluorescence intensity ofeach well (black bottom 96-well plate) was measured by a Tecan platereader with λ_(Ex)=321 nm and λ_(Em)=445 nm and data was normalized tothe value of pH 2.5. Typically, the apparent pK_(a) is defined by the pHat half-maximum fluorescence. Although this method was useful forestimating LNP global/apparent pK_(a) for most all LNPs, it could not beused for SORT LNPs containing >40% permanently cationic lipid becausethese LNPs are always charged. Therefore, the relative pK_(a) wasinstead estimated compared to base LNP formulation (no added SORT lipid)when 50% of normalized signal was produced. This alternative calculationdid not change the pK_(a) for most LNPs but did allow estimation ofpermanently cationic SORT LNPs that agreed with experimental results fortissue selective RNA delivery. Thus, it can be suggested that thestandard TNS assay be used when LNPs contain a single ionizable cationiclipid and the alternative 50% normalized signal method be used forsystems such as SORT that contain complex mixtures of multiple lipidsharboring a variety of charge states.

IV. In Vitro Luciferase Expression and Cell Viability Tests

Huh-7 or A549 cells were seeded into white 96-well plates at a densityof 1×10⁴ cells per well the day before transfection. The media wasreplaced by 150 μL fresh DMEM medium (5% FBS), then 50 μL Luc mRNAformulations were added with fixed 25 ng mRNA per well. After incubationfor another 24 h, ONE-Glo+Tox kits were used to detect mRNA expressionand cytotoxicity based on Promega's standard protocol.

V. Animal Experiments

All animal experiments were approved by the Institution Animal Care andUse Committees of The University of Texas Southwestern Medical Centerand were consistent with local, state and federal regulations asapplicable. C57BL/6 mice were obtained from the UTSW Mouse Breeding CoreFacility. B6.Cg-Gt(ROSA)26Sor^(tm9(CAG-tdTomato)Hze)/J mice (also knownas Ai9 or Ai9(RCL-tdT) mice) were obtained from The Jackson Laboratory(007909) and bred to maintain homozygous expression of the Cre reporterallele that has a loxP-flanked STOP cassette preventing transcription ofa CAG promoter-driven red fluorescent tdTomato protein. FollowingCre-mediated recombination, Ai9 mice will express tdTomato fluorescence.Ai9 mice are congenic on the C57BL/6J genetic background.

VI. In Vivo Luc mRNA Delivery and Biodistribution

C57BL/6 mice with weight of 18-20 g, were IV injected by various LucmRNA formulations at a dose of 0.1 or 0.05 mg/kg. n=2-4 per group. After6 h, mice were intraperitoneal (IP) injected with D-Luciferin (150mg/kg) and imaged by an IVIS Lumina system (Perkin Elmer). Forbiodistribution, C57BL/6 mice were IV injected with Cy5-Luc mRNAformulations at a dose of 0.5 mg/kg. Ex vivo imaging (Cy5 channel) wasperformed 6 h post injection.

VII. mRNA Synthesis

Optimized Cre recombinase mRNA and Cas9 mRNA was produced by in vitrotranscription (IVT). Briefly, NLS-Cre fragment and Cas9 fragment wereprepared by PCR program using pCAG-CreERT2 and pSpCas9(BB)-2A-GFP(PX458) as PCR template, respectively. Then, the these fragments werecloned into pCS2+MT vector with optimized 5′(3′)-untranslated regions(UTR) and poly A sequences. IVT reactions were performed followingstandard protocols but with N1-methylpseudouridine-5′-triphosphatereplacing the typical UTP. Finally, the mRNA was capped (Cap-1) byVaccinia Capping Enzyme and 2′-O-methyltransferase (NEB). Table 4 showsthe primers used herein.

TABLE 4  Primers including the length of PCR products and their purposesForward Primers  Reverse Primers  Name (5′ to 3′) (5′ to 3′) LengthNotes Cas9 ATATATGGATCCGCCACC ATATATGAATTCTTA 4233 bp For IVTATGGCCCCAAAGAAGAAG CTTTTTCTTTTTTGC clone CGGAAGGTC CTGGCCGGCCTTTTC(SEQ ID NO: 3) GTGGCCGCCGGCCTT TTGTCGCCTCCCAG (SEQ ID NO: 4) Ca9 Seq-1CTGAGCGACATCCTGAGA For  GTGAAC  sequencing (SEQ ID NO: 5) to confirmCa9 Seq-2 AGCAGGTCCTCTCTG the whole TTCAG Cas9 (SEQ ID NO: 6) sequencesCa9 Seq-3 GACGGCTTCGCCAACAGA AACTTC (SEQ ID NO: 7) Ca9 Seq-4TTTGATGCCCTCTTC GATCCG (SEQ ID NO: 8) Ca9 Seq-5 GGGAGATCGTGTGGGATAAG (SEQ ID NO: 9) Ca9 Seq-6 ACTTCTTAGGGTCCC AGTCC (SEQ ID NO: 10)Ca9 Seq-7 AAGAGAGTGATCCTGGCC GAC (SEQ ID NO: 11) NLS-CreATATATGGATCCGCCACC ATATATGAATTCTTA 1083 bp For IVT ATGCCCAAGAAGAAGAGGATCGCCATCTCCAGC clone AAGGTGGCCAATTACTGA AG CCGTACACCAAAATTTGC(SEQ ID NO: 13) CTG (SEQ ID NO: 12) PTEN ATCCGTCTTCTCCCCATTGACGAGCTCGCTAAT  638 bp For T7E1 CCG (SEQ ID NO: 14) CCAGTG assay(SEQ ID NO: 15)

The code sequences for NLS-Cre and Cas9 as following:

NLS-Cre: (SEQ ID NO: 16)ATGCCCAAGAAGAAGAGGAAGGTGGCCAATTTACTGACCGTACACCAAAATTTGCCTGCATTACCGGTCGATGCAACGAGTGATGAGGTTCGCAAGAACCTGATGGACATGTTCAGGGATCGCCAGGCGTTTTCTGAGCATACCTGGAAAATGCTTCTGTCCGTTTGCCGGTCGTGGGCGGCATGGTGCAAGTTGAATAACCGGAAATGGTTTCCCGCAGAACCTGAAGATGTTCGCGATTATCTTCTATATCTTCAGGCGCGCGGTCTGGCAGTAAAAACTATCCAGCAACATTTGGGCCAGCTAAACATGCTTCATCGTCGGTCCGGGCTGCCACGACCAAGTGACAGCAATGCTGTTTCACTGGTTATGCGGCGTATCCGAAAAGAAAACGTTGATGCCGGTGAACGTGCAAAACAGGCTCTAGCGTTCGAACGCACTGATTTCGACCAGGTTCGTTCACTCATGGAAAATAGCGATCGCTGCCAGGATATACGTAATCTGGCATTTCTGGGGATTGCTTATAACACCCTGTTACGTATAGCCGAAATTGCCAGGATCAGGGTTAAAGATATCTCACGTACTGACGGTGGGAGAATGTTAATCCATATTGGCAGAACGAAAACGCTGGTTAGCACCGCAGGTGTAGAGAAGGCACTTAGCCTGGGGGTAACTAAACTGGTCGAGCGATGGATTTCCGTCTCTGGTGTAGCTGATGATCCGAATAACTACCTGTTTTGCCGGGTCAGAAAAAATGGTGTTGCCGCGCCATCTGCCACCAGCCAGCTATCAACTCGCGCCCTGGAAGGGATTTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGATGACTCTGGTCAGAGATACCTGGCCTGGTCTGGACACAGTGCCCGTGTCGGAGCCGCGCGAGATATGGCCCGCGCTGGAGTTTCAATACCGGAGATCATGCAAGCTGGTGGCTGGACCAATGTAAATATTGTCATGAACTATATCCGTAACCTGGATAGTGAAACAGGGGCAATGGTGCGCCTGCTG GAAGATGGCGATTAA SV40 NLS-Cas9-Nucleoplasmin NLS: (SEQ ID NO: 17)ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGTAA

VIII. Western Blot

The quality of IVT Cas9 mRNA was analyzed by western blot. 293T cellswere seeded into 12-well plate with the density of 1×10⁵ cells per wellthe day before transfection. Cells were treated with variousformulations in 600 μL total volume for another 24 h, including mCherrymDLNP (0.5 μg mRNA per well), mCherry mDLNP (1.0 μg mRNA per well), IVTCas9 mDLNP (0.5 μg mRNA per well), IVT Cas9 mDLNP (1.0 μg mRNA per well)and Lipofectamine2000/Cas9 pDNA (0.5 μg pDNA per well). After washingthree times with 1×PBS, 100 μL lysis buffer (50 mM Tris HCl, pH 7.4,with 150 mM NaCl, 1 mM EDTA and 1% TRITON X-100) and 1 μL proteininhibitor cocktail (100×, Thermo Fisher) were added into each well androcked for 20 min at RT. Cell lysates were collected into 1.6 mL tubesand centrifuged for 10 min (13,000 g) at 4° C. Supernatants werecollected into new tubes and stored in −80° C. if not used immediately.Before executing a western blot, protein concentrations were measuredusing a BCA assay kit (ThermoFisher). Fifteen microgram total proteinswere loaded and separated by 4-20% polyacrylamide gel (ThermoFisher).Separated proteins were then transferred into polyvinylidene membrane(BioRad) and blocked by 5% BSA (dissolved in PBST) for 1 h at RT.Primary antibodies were applied overnight at 4° C. After washing fourtimes using PBST, the membrane was incubated by secondary antibody for 1h at RT then imaged with ECL substrate after washing four times by PBST(ThermoFisher).

IX. Gene Editing (Cre mRNA) in Td-Tomato Mice Model

Cre mRNA formulations were prepared as described above and performed IVinjections (0.3 mg/kg Cre mRNA). After two days, mice (n=4 per group)were sacrificed and major organs were imaged by IVIS Lumina system(Perkin Elmer).

X. Cell Isolation and Staining for Flow Cytometry

To test the Td-Tomato⁺ cells in cell types of each organ, cell isolationand staining was performed after 2 days of treatment with Cre mRNAformulations (0.3 mg/kg), then analyzed by flow cytometry.

For hepatocyte isolation, two-step collagenase perfusion was executed asdescribed before (Cheng et al, 2018). Briefly, mice were anesthetized byisofluorane and fixed. Perfusion was started with liver perfusion medium(Thermo Fisher Scientific, 17701038) for 7-10 min, then switched toliver digestion medium (Thermo Fisher Scientific, 17703034) for another7-10 min. The liver was collected into a plate containing 10 mL of liverdigestion medium and cut to release the hepatocytes. Then the releasedhepatocytes were collected and washed twice with hepatocyte wash medium(Thermo Fisher Scientific, 17704024) and once with 1×PBS. After furtherisolated by straining and low speed (50×g) centrifugation, thehepatocytes were analyzed by FACS Aria II SORP machine (BD Biosciences).

For isolation and staining of spleen cell types, the removed spleen wasminced up by a sterile blade and homogenized in 250 μL of 1× digestionmedium (45 units/μL Collagenase I, 25 units/μL DNAse I and 30 units/μLHyaluronidase). The spleen solution was transferred into a 15 mL tubethat contained 5-10 mL of 1× digestion medium. Next, the spleen solutionwas filtered using a 70 μm filter and washed once with 1×PBS. A cellpellet was obtained by centrifuging for 5 min at the speed of 300×g. Thesupernatant was removed and the cell pellet was resuspended in 2 mL of1×RBC lysis buffer (BioLegend, 420301) and incubated on ice for 5 min.After incubation, 4 mL of cell staining buffer (BioLegend) was added tostop RBC lysis. The solution was centrifuged again at 300×g for 5 min toobtain a cell pellet. The single cells were resuspended in cell stainingbuffer and added into flow tubes that contained antibodies (100 μL totalvolume). The cells were incubated with antibodies for 20 min in the darkat 4° C. The stained cells were washed twice with 1 mL 1×PBS, thenresuspended in 500 μL 1×PBS for flow cytometry analysis. The antibodiesused were Pacific Blue anti-mouse CD45 (BioLegend, 103126), Alexa Fluor488 anti-mouse/human CD11b (BioLegend, 101217), Alexa Fluor 647anti-mouse CD19 (BioLegend, 115522) and PerCP-Cyanine5.5 Anti-Mouse CD3e(145-2C11) (Tonbo Biosciences, 65-0031). Ghost Dye Red 780 (TonboBiosciences, 13-0865-T500) was used to discriminate live cells.

For isolation and staining of lung cell types, isolated lungs wereminced up by a sterile blade and then transferred into 15 mL tube thatcontained 10 mL 2× digestion medium (90 units/μL Collagenase I, 50units/μL DNAse I and 60 units/μL Hyaluronidase) and incubated at 37° C.for 1 h with shaking. After incubation, any remaining lung tissue washomogenized. The following steps were similar with the spleen protocoldescribed above. The antibodies here used were Pacific Blue anti-mouseCD45 (BioLegend, 103126), Alexa Fluor 488 anti-mouse CD31 (BioLegend,102414) and Alexa Fluor 647 anti-mouse CD326 (Ep-CAM) (BioLegend,118212). Ghost Dye Red 780 (Tonbo Biosciences, 13-0865-T500) was used todiscriminate live cells.

XI. Gene Editing (Cas9 mRNA/sgRNA and Cas9/sgRNA RNPs) in Td-Tomato MiceModel

To evaluate in vivo gene editing, Td-Tom mice were selected forcomparable weight and same sex. Cas9 mRNA and sgRNA was co-delivered totdTomato (td-Tom) mice. Cas9 mRNA/sgTom1 (4/1, wt/wt) were co-deliveredby various formulations with the total RNA dose equal to 2.5 mg/kg. 10days following IV injection, the main organs were removed and imaged onan IVIS Lumina system. For spleen-targeted formulations, the total RNAdose was 4 mg/kg and the weight ratio of Cas9 mRNA to sgTom1 was 2/1,with a detection time was 2 days. For RNP delivery, the mole ratio ofCas9 protein to sgRNA was fixed at 1:3, the injection dose was 1.5 mg/kgRNA, and the detection time was day 7 after injection (n=2-4 per group).To confirm Td-Tom expression, tissue sections were further prepared andimaged by confocal microscopy. Briefly, tissue blocks were embedded intooptimal cutting temperature compound (OCT) (Sakura Finetek) andcyro-sectioned (8 μm) on a Cryostat instrument (Leica Biosystems).Mounted tissue slices were stained with 4,6-diamidino-2-phenylindole(DAPI, Vector Laboratories) before imaging by confocal microscopy on aZeiss LSM 700.

XII. Gene Editing (Cas9 mRNA/sgPTEN and Cas9/sgRNA RNPs) in C57BL/6 Mice

To examine endogenous gene editing in vivo, PTEN was selected. Wild typeC57BL/6 mice were IV injected with various carriers by co-delivery ofCas9 mRNA and modified sgPTEN at a total dose of 2.5 mg/kg (4/1,mRNA/sgRNA, wt/wt) (n=2-4 per group). After 10 days, tissues werecollected, and genomic DNA was extracted using a PureLink Genomic DNAMini Kit (ThermoFisher). For spleen-targeted formulations, the total RNAdose was 4 mg/kg, Cas9 mRNA/sgTom1 was 2/1 (wt/wt), and the detectiontime was 2 days after injection. For RNP delivery, the mole ratio ofCas9 protein to sgRNA was fixed at 1:3, the injection dose was 1.5 mg/kgRNA, and the detection time was day 7 after injection (n=2-4 per group).After obtaining PTEN PCR products, the T7E1 assay (NEB) was performed toconfirm gene editing efficacy by the standard protocol. Furthermore,evaluation of PTEN editing was executed on tissue sections by H&Estaining and immunohistochemistry (IHC). Briefly, paraformaldehyde (PFA)fixed tissues were embedded in paraffin, sectioned and H&E stained bythe Molecular Pathology Core at UTSW. The 4 μm sections were performedin the standard fashion and detected with Elite ABC Kit and DABSubstrate (Vector Laboratories) for IHC.

Example 9: Formulation Using Neutral Buffer

Cas9 RNPs was observed to denature in acidic buffer, resulting in anincrease of hydrodynamic size from 10 nm to 150 nm (FIG. 40B). Thismakes RNP encapsulation into monodisperse nanoparticles difficult if notimpossible. These studies focus on lipid nanoparticles (LNPs) becausethey are the most efficacious class of RNA delivery carriers (Wang etal., 2017; Doudna & Charpentier, 2014; Hajj & Whitehead, 2017; Sander &Joung, 2014) in preclinical models and in humans (Wood, 2018). Among thefour components of LNPs [ionizable cationic lipids, zwitterionicphospholipids, cholesterol, and poly(ethylene glycol) (PEG) lipids)],ionizable cationic lipids with pK_(a) around 6.4 are useful for activitybecause they bind negatively charged RNAs at the pH of mixing (e.g. pH 4when the amines are protonated), lose charge at neutral pH beforecellular uptake, and then acquire charge again as the pH in endosomesdecreases to fuse with endosomal membranes and enable release of cargoesto the cytoplasm. However, this feature prevents effective encapsulationof cargoes at neutral pH because ionizable cationic lipids are unchargedat neutral pH. To overcome this challenge, the addition of a 5^(th)component, specifically a cationic lipid that would be positivelycharged at neutral pH, would allow for encapsulation of RNAs andproteins using neutral buffers (instead of acidic buffers), thuspreserving the tertiary structure and stability of RNPs (FIG. 40A).

To evaluate this strategy, 5A2-SC8 was selected as the ionizablecationic lipid because 5A2-SC8 LNPs safely deliver short siRNAs/miRNAsand long mRNAs to mice with compromised liver function, includingMYC-driven liver cancer (Zhou et al., 2016; Zhang et al., 2018a; Zhanget al., 2018b) or genetic knockout of fumarylacetoacetate hydrolase(FAH) (Cheng et al., 2018). Introduction of a permanently cationic lipid(e.g. DOTAP) into traditional 4-component 5A2-SC8 LNP formulationsindeed allowed controlled self-assembly to occur by mixing an ethanolsolution of lipids with a PBS solution of RNPs (1/3, v/v). Theincorporation of DOTAP was evaluated from 5 to 60 mole % of total lipids(FIG. 41), which revealed higher levels of gene editing at 10-20% invitro and formation of stable RNP-loaded nanoparticles with size <200 nm(FIG. 42). Initially using sgRNA targeting reporter Luciferase (sgLuc),the size of LNPs with 10 mole % DOTAP incorporation (5A2-DOT-10:5A2-SC8/DOPE/Chol/DMG-PEG/DOTAP=15/15/30/3/7 (mol/mol)) was observed,prepared using PBS buffer were slightly larger than nanoparticleswithout RNP loading. Identical LNPs prepared using low PH buffer did notchange size, implying that RNPs were not encapsulated (FIG. 40C). Todetermine the optimal mole ratio between Cas9 protein and sgRNA,Cas9/sgRNA complexes at 1/1, 1/3 and 1/5 (mol/mol) were prepared, whichdecreased RNP size (FIG. 40D) and increased negative charge (FIG. 40F).The RNP ratio did not alter the size or zeta potential of the resultingLNPs after encapsulation (FIG. 1E, 1G). The surface charge of all LNPswas neutral, which not only indicates successful encapsulation, but isalso useful for minimizing in vivo uptake by the immune mononuclearphagocyte system (MPS) system. To further examine whether 5A2-DOT-10could successfully mediate delivery of RNPs into the nucleus, LNPs withencapsulated fluorescent EGFP-fused Cas9 protein were tracked. Free RNPsalone were unable to enter cells, as no green fluorescence abovebackground was detectable (FIG. 43). Bright green fluorescence wasobserved in the cytoplasm of cells after 5A2-DOT-10 treatment for 3hours. EGFP-fused Cas9 proteins were then observed to gradually enterthe nucleus within 6 hours (FIG. 40H) due to the presence of nuclearlocalization signals on Cas9. Endocytosis was energy dependent andmainly dependent on lipid rafts, as treatment of MβCD, an inhibitor oflipid raft-based endocytosis, significantly inhibited cellular uptake ofnanoparticles (FIG. 40I).

To quantify gene editing efficacy, HeLa-Luc and HeLa-GFP reporter cellswere employed. Examining different Cas9/sgLuc ratios, gene editing washigher at 1/3 and 1/5 (FIG. 44A). The result of a T7 Endonuclease I(T7EI) assay demonstrated that most all target DNA bands (720 bp) werecut into two cleavage bands (536 bp and 184 bp). No cleavage bands wereobserved with control treatment groups. To test the hypothesis thatneutral pH buffer is required to encapsulate RNPs with preservation ofCas9 function, the gene editing efficiency of 5A2-DOT-10 prepared usingpH 4 citrate buffer was also evaluated. No cleavage bands were observedat all (FIG. 44A). The negative result was further confirmed by Sangersequencing, providing additional evidence that the conventionalacid-based formulation methods could not produce efficacious NPs.Switching to GFP-expressing cells, 5A2-DOT-10 encapsulating Cas9/sgGFPinduced Indels into GFP DNA and knocked out nearly all GFP expression.Control groups exhibited similar fluorescence intensity with PBS-treatedcells (FIG. 44B), which was confirmed by flow cytometry (FIGS. 44C &45). Permanent gene editing was apparent by an indefinite loss of GFP ingrowing cells and confirmed by Sanger sequencing, where Inference ofCRISPR Edits (ICE) analysis showed that indels reached 95% (FIG. 44D).With an eye towards clinical translation, RNP-loaded 5A2-DOT-10stability was monitored at 4° C. for 2 months. LNPs did not change sizeand remained uniform (PDI<0.2) (FIG. 44G). Continual testing of5A2-DOT-10 nanoparticles revealed constant gene editing activity, evenafter 60 days storage (FIG. 44H).

The strategy of adding a permanently cationic lipid into classical4-component LNPs to achieve efficient RNP delivery was not limited tothe dendrimer-based ionizable lipid, 5A2-SC8. To prove this,supplemental DOTAP was included into nanoformulations prepared usingother classes of ionizable materials: the well-known DLin-MC3-DMA lipidused in FDA-approved Onpattro (Wood, 2018) and the C12-200 lipidoid(FIGS. 46A-B). Even though they have very different chemical structurescompared to 5A2-SC8 (FIG. 46C), all DOTAP-modified nanoparticles couldefficiently edit cells whereas previously established C12-200 or MC3formulations without DOTAP showed low editing efficiency (FIG. 44E).5A2-DOT-10 also achieved higher editing efficiency than the positivecontrol RNAiMAX. Because 5A2-DOT-10 LNPs were more efficacious thanMC3-DOT-10 and C12-200-DOT-10, all subsequent experiments were performedusing 5A2-SC8. In addition to DOTAP, other cationic lipids, includingDDAB and EPC, were also introduced into LNP formulations (FIGS. 46E-G).The results were similar for all three cationic lipids with differentchemical structures (FIG. 46H). These results indicate that thisstrategy is universal for ionizable cationic lipid nanoparticles (DLNPs,LLNPs, SNALPs) and for other cationic lipids that are positively chargedat pH 7.4. Because this methodology allowed adjustment of theFDA-approved Onpattro formulation to enable delivery of RNPs, thisapproach offers different directions for clinically translatabletreatment of human diseases.

A key to successful RNP delivery is replacement of the standard acidicbuffer with PBS buffer to maintain protein stability. To test if thismethodology is compatible with other neutral buffers, LNPs in PBS,Opti-MEM medium, and HEPES were formulated. Formulations prepared incitrate buffer (pH 4) were used as a control (FIG. 46I). Significant andequivalent gene editing (>90%) was achieved using LNPs prepared in allthree neutral buffer conditions, but not in acidic buffer (FIG. 44F).ICE analysis of sequencing results was consistent with that shown byflow cytometry (FIG. 46K).

To examine in vivo gene editing, 5A2-DOT-10 encapsulating Cas9/sgTOMcomplexes to the Td-Tomato mouse model (FIG. 47A) were delivered. Inthese mice, CRISPR-mediated deletion of the Lox-Stop-Lox cassette turnson downstream tdTom expression in successfully edited cells. 5A2-DOT-10LNPs loaded with Cas9/sgTOM RNPs were injected into the left leg of miceby intramuscular injection at dose of 1 mg/kg sgTOM. Due to previous usefor direct injection gene editing, (Zuris et al., 2015) RNAiMAXcomplexed with Cas9/sgTOM RNPs was used for comparison. Higher Td-Tomfluorescence was observed in the muscle treated with 5A2-DOT-10 than inmice treated with RNAiMAX (FIG. 47B). Imaging of tissue sections furtherconfirmed gene editing producing brighter red fluorescence in the5A2-DOT-10 treatment group (FIG. 47C). 5A2-DOT-10 were injected into thebrains of Td-Tom mice (0.15 mg/kg of sgTOM). Again, bright red signalwas observed near the injection site, confirming editing of mouse brains(FIG. 47D-E).

The improved stability and efficacy of 5A2-DOT-10 could mediatesuccessful systemic gene editing in tissues were evaluated. To examinethis strategy for RNP delivery, LNPs were prepared with different molarpercentages of DOTAP (5-60%) and delivered RNPs to Td-Tom mice IV (1.5mg/kg of sgTOM). Td-Tom fluorescence was observed exclusively in theliver 7 days following injection of 5A2-DOT-5. Increasing theincorporated DOTAP percentage from 5 to 60% resulted in gradualfluorescence (CRISPR-guided gene editing) from liver to lung. 5A2-DOT-60enabled mainly lung editing (FIG. 47F). These results indicate that deeptissue editing can be achieved in a tissue-specific manner by adjustingthe inner lipid component chemistry and molar ratios. Tissue-specificediting was further confirmed by confocal imaging of tissue sections(FIG. 47G). The editing of an endogenous target, Pten, were thenevaluated by systemically injecting LNPs encapsulating Cas9/sgPTEN RNPsinto wild-type C57BL/6 mice. Clear T7EI cleavage bands were onlydetected in liver for 5A2-DOT-5 treated mice and in the lungs for5A2-DOT-50 and 5A2-DOT-60 treated mice (FIG. 47H).

To evaluate whether it is possible to simultaneously edit multiple genesin vivo, Cas9 protein and six different sgRNAs into 5A2-DOT-50. sgTOM,sgP53, sgPTEN, sgEml4, sgALK and sgRB1 were loaded into Cas9 proteinswere encapsulated. Td-Tom mice with 5A2-DOT-50 (Pool) by tail veininjection (0.33 mg/kg of each sgRNA) were then treated. After one week,bright Td-Tom fluorescence was detected in the lungs, indicating geneediting of TOM (FIG. 47I). Clear T7EI cleavage bands were observed atall other 5 genome loci, demonstrating 5A2-DOT-50 were able to editmultiple genes simultaneously and effectively (FIG. 47J) at low doses.Quantification analyses revealed editing efficiencies of targets up to22% (FIGS. 47 & 48) in the lungs. The sgRNAs with end modifications ofthe first and last 3 nucleotides were used herein to enhance sgRNAstability and reproducibility (FIG. 49) (Finn et al., 2018; Hendel etal., 2015). Reports have shown that precise modifications to additionalnucleotides can increase in vivo gene editing 2- to 4-fold compared toend-modified sgRNAs, (Finn et al., 2018; Yin et al., 2017) suggestingthat the editing efficiencies reported herein could be higher withfurther sgRNA optimization. Nevertheless, the high potency and tissuespecificity of 5A2-DOT-50 allowed for simultaneous editing of 6 targetsin the lungs with one injection.

Animal models are traditionally generated by transgenesis or geneengineering in embryonic stem cells, which is time consuming and costly.Direct mutation of tumor- and other disease-related genes in adult miceusing CRISPR/Cas provides a feasible approach for rapid generation ofmodels. This has only been accomplished using costly lentiviruses thatmust be engineered for each target and by hydrodynamic injection intothe liver (Xue et al., 2014; Maddalo et al., 2014). Since mutation ofmultiple genes is typically required to generate functional cancermodels, the development of an inexpensive and effective non-viralnanoparticle-based approach for multiplexing is highly desirable. Since5A2-DOT-X LNPs are potent, can simultaneously edit multiple targets, canbe administered repeatedly, and provide tissue specificity, they providea path to generate a wide variety of animal models.

5A2-DOT-5 were employed to simultaneously knockout three tumorsuppressor genes (P53, PTEN, and RB1) selectively in the liver. Thesegenes have been identified in many human cancers, including liver.C57BL/6 mice were treated with weekly IV injections of 2.5 mg/kg totalsgRNA for 3 weeks and detected the gene editing efficiency in micelivers (FIG. 48A). The clear cleavage bands at all three gene loci wereobserved after treatments of 2, 12, 15, and 20 weeks by T7EI assay(FIGS. 48B, 50, & 51). The cleavage bands were much brighter as timeprogressed, indicating tumor growth. When the mice were sacrificed at 15weeks and 20 weeks, visible tumors were found on the liver, togetherwith several metastatic tumors in the abdominal cavities (FIGS. 48C &52). The tumor generation by H&E staining and IHC staining targetingtumor proliferation biomarker Ki67 (FIGS. 48D & 53) were also detectedat various time points.

To generate a challenging lung cancer mouse model, the Eml4-Alkchromosomal rearrangement was focused on, which is a complex mutationfound in many solid human tumors, especially non-small cell lung cancers(Maddalo et al., 2014; Blasco et al., 2014). The Eml4-Alk fusion proteingenerated after rearrangement between Eml4 and Alk promotes cancerdevelopment. Exploiting the high potency and lung-targeting specificityof 5A2-DOT-50, once (at dose of 2 mg/kg of total sgRNA) or twice (atdose of 1.5 mg/kg of total sgRNA, weekly) IV doses were injected andevaluated the tumor generation process (FIG. 48E). Indel generation wasdetectable at all examined time points from extracted lung DNA from micein both groups (FIGS. 48F & 54). Clear gene rearrangement bands weredetected in the lungs of 5A2-DOT-50-treated mice, confirmingsuccessfully generated chromosomal rearrangements (FIGS. 48F & 54). TheEml4-Alk rearrangement bands were much brighter as time progressed,suggesting proliferation of edited cells. The sequencing results aftersub-cloning of these PCR amplicons further confirmed the Eml4-Alkrearrangements (FIGS. 48G & 54). Several tumor lesions were observed inthe lungs after 16 weeks and 24 weeks from H&E staining and Ki67staining (FIGS. 48H, 55, & 56). These results show that a singleinjection of 5A2-DOT-50 LNPs could successfully generate chromosomalrearrangements and lead to lung tumor generation in adult mice. TheseLNPs are therefore positioned to accelerate in situ creation of avariety of disease models.

B. Materials and Methods

I. Materials.

5A2-SC8 (Zhou et al., 2016), DLin-MC3-DMA (Jayaraman et al., 2012), andC12-200 (Love et al., 2010) were synthesized and purified by followingpublished protocols. 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine(DOPE), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),dimethyldioctadecylammonium (DDAB),1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), and1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were purchased fromAvanti Polar Lipids. Cholesterol was purchased from Sigma-Aldrich.1,2-Dimyristoyl-sn-glycerol-methoxy(poly((ethylene glycol) MW 2000)(DMG-PEG2000) was purchased from NOF America Corporation. TheONE-Glo+Tox Luciferase Reporter assay kit was purchased from PromegaCorporation. Pur-A-Lyzer Midi Dialysis Kits (WMCO, 3.5 kDa) werepurchased from Sigma-Aldrich. 4′,6-Diamidino-2-phenylindoledihydrochloride (DAPI), Hoechst 33342, DLS Ultramicro cuvettes,Lipofectamine RNAiMAX Transfection Reagent, and Lab-Tek chambered coverglass units were purchased from Thermo Fisher Scientific. Cas9 proteinand Ki-67 monoclonal antibody was purchased from Thermofisher. GenCrisprNLS Cas9-EGFP Nuclease was purchased from GenScript. Modified sgRNAswere purchased from Synthego.

II. Cas9/sgRNA Complex Preparation.

Separate solutions of Cas9 proteins and sgRNAs in the notated bufferswere mixed together at equal volumes. After mixing, the RNPs wereallowed to form over 5 minutes of incubation at room temperature forfull Cas9/sgRNA complex self-assembly. The mole ratios of Cas9 proteinto sgRNA used were 1/1, 1/3 and 1/5.

III. Optimized Nanoparticle Formulations and Characterization.

Ionizable cationic lipids (5A2-SC8, C12-200, or DLin-MC3-DMA) (Zhou etal., 2016; Jayaraman et al., 2012; Love et al., 2010), zwitterioniclipids (DOPE or DSPC), cholesterol, DMG-PEG, and permanently cationiclipids (DOTAP, DDAB, or EPC) were dissolved in ethanol at given molarratios. Cas9/sgRNA complexes were dissolved in 1×PBS buffer. TheCas9/sgRNA RNP complexes solution in PBS buffer was pipette mixedrapidly into the lipids solution in ethanol at a volume ratio of 3:1(Cas9/sgRNA RNPs:lipids, v/v), such that the weight ratio of totallipids to sgRNA was 40:1 (wt), then incubated for 15 min at roomtemperature. Afterwards, the fresh formulations were directlycharacterized and used for in vitro assays. For animal experiments, theformulations were dialyzed (Pur-A-Lyzer Midi Dialysis Kits, WMCO 3.5kDa) against 1×PBS for 1 h to remove ethanol before topical injections(intra-muscle or intra-brain injection) or systemic injection(intravenous injection). The size distribution and zeta potential ofnanoformulations were measured using Dynamic Light Scattering (DLS,Malvern; He—Ne Laser, k=632 nm; detection angle=173°).

IV. RNAiMAX Formulations.

For preparation of RNAiMAX complexing RNPs, the Cas9/sgRNA complex wasprepared in Opti-MEM and mixed gently with Lipofectamine RNAiMAXTransfection Reagent diluted in Opti-MEM (at dose of 1 μL RNAiMAX/μgsgRNA). The mixture solution was incubated at room temperature for 30minutes to complete the complexation.

V. Standard LNP Formulations.

For preparation of C12-200 and MC3 LNPs encapsulating RNPs, Cas9/sgRNARNP complexes solution in citrate buffer (pH 4.0) was pipette mixedrapidly into the lipids solution in ethanol at a volume ratio of 3:1(Cas9/sgRNA RNPs:total lipids, v/v), such that the weight ratio of totallipids to sgRNA was 40:1 (wt/wt), then incubated for 15 min at roomtemperature. The molar ratio of C12-200/DOPE/Chol/DMG-PEG was35/16/46.5/2.5 for C12-200 LNPs; the molar ratio ofDLin-MC3-DMA/DSPC/Chol/DMG-PEG was 50/10/38.5/1.5 for MC3 LNPs.

VI. Cellular Uptake and Uptake Mechanism of 5A2-DOT-10 Cas9/sgLucTreatment.

To examine cellular uptake, HeLa-Luc cells were seeded into Lab-TekChambered Coverglass (8 wells) at a density of 2×10⁴ cells per well andincubated at 37° C. overnight. Then, the old media was replaced with 150μL of fresh DMEM containing 10% FBS and treated with 50 μL of 5A2-DOT-10encapsulating Cas9-EGFP/sgLuc RNPs (9 nM of sgRNA per well). At 1 h, 3h, 6 h, and 24 h after treatment, cells were washed three times with PBSand stained with Hoechst (0.1 mg/mL) for 15 min at 37° C., then imagedby confocal microscopy (Zeiss LSM 700).

To examine the uptake mechanism, assays of specific inhibition onendocytosis pathways were evaluated using Hela-Luc cells. 5A2-DOT10 onlytreatment was used as a control. HeLa-Luc cells were seeded at a densityof 5×10⁵ cells per well in 12-well plates and incubated in DMEM completemedium for 24 h. The cells were then washed with PBS and followed bypre-incubating at 37° C. for 1 h with one of the following endocytosisinhibitors dissolved in Opti-MEM: 20 μM chlorpromazine (CMZ, aninhibitor of clathrin-mediated endocytosis), 2 mM Amiloride (AMI, aninhibitor of macropinocytosis), 200 μM Genistein (GEN, an inhibitor ofcaveolae-mediated endocytosis), 5 mM Methyl-o-cyclodextrin (MPCD, aninhibitor of lipid rafts-mediated endocytosis). Next, the medium wasremoved and replaced with complete DMEM medium containing 5A2-DOT-10Cas9/sgLuc (24 nM sgLuc) for another 30 min. After that, the medium wasremoved and the cells were washed three times with PBS. The cells werethen collected and analyzed by flow cytometry. All experiments werecarried out in triplicate. Here, Cas9-EGFP protein was used to formulateCas9/sgLuc complex. To evaluate whether it is energy dependentendocytosis, the cells were also pre-incubated under 4° C. for 1 h, andthen treated with complete DMEM medium containing 24 nM of 5A2-DOT-10Cas9/sgLuc (24 nM sgLuc) for another 30 min before analysis by flowcytometry.

VII. T7EI Assay to Detect Genomic Editing.

For in vitro genomic DNA editing analysis, HeLa-Luc cells were seededinto 12-well plates at a cell density of 1.5×10⁵ cells/well andincubated overnight. Then, different nanoformulations containing 24 nMof sgRNA were added to cells. After 3 days, the cells were collected,washed and re-suspended in 50 μL of 1× passive lysis buffer (Promega)together with 2 μL of proteinase K (Thermofisher). Afterwards, a lysisPCR program (65° C. for 15 min, 95° C. for 10 min) was run to obtaincell lysates. The targeted genomic loci were then amplified using thefollowing PCR amplification program (95° C. for 5 min; (95° C. for 30sec; 60-64° C. for 30 sec; 72° C. for 1 min) for 40 cycles; 72° C. for 7min and then keep at 4° C.). Cell lysates were used as DNA templates.The amplicons were then purified using PCR purification kits (Qiagen)and 200 ng of the purified DNA was added to 19 μL of annealing reactioncontaining 1×NEBuffer 2. Then the PCR products were annealed in athermocycler using the following conditions (95° C. for 5 min, then themixture was cooled from 95° C. to 85° C. with Ramp Rate of −2°C./second, following 85° C. to 25° C. with Ramp Rate of ˜0.1° C./second,then keep at 4° C.) to form heretoduplex DNA. Afterwards, 1 μL of T7EI(NEB) was added and incubated at 37° C. for 15 min. The cleavagereaction was then stopped by adding 1.5 uL of 0.25M EDTA. Next, thedigested DNA was analyzed using 2.5% agarose gel electrophoresis. Allprimers used for T7EI assay are listed in Table 5.

TABLE 5  sgRNA Sequences Name Target Sequences (5′ to 3′) PAM (5′ to 3′)sgLUC CTTCGAAATGTCCGTTCGGT TGG (SEQ ID NO: 18) sgGFPGAAGTTCGAGGGCGACACCC TGG (SEQ ID NO: 19) sgTOM AAGTAAAACCTCTACAAATG TGG(SEQ ID NO: 20) sgPTEN AGATCGTTAGCAGAAACAAA AGG (SEQ ID NO: 21) sgP53GTGTAATAGCTCCTGCATGG GGG (SEQ ID NO: 22) sgRB1 TCTTACCAGGATTCCATCCA CGG(SEQ ID NO: 23) sgEm14 CCTGCCCTGAGTAAGCGACA CGG (SEQ ID NO: 24) sgAlkTCCTGGCATGTCTATCTGTA AGG (SEQ ID NO: 25)

For in vivo genomic DNA editing analysis, genomic DNA was extracted fromtissues using PureLink Genomic DNA Mini Kit (Invitrogen) according tomanufacturer's instructions. Subsequently, the aforementioned procedureswere followed as described above for T7EI detection.

The fragmented PCR products were analyzed and the indels percentageswere calculated based on the following formula:% gene modification=100×(1−(1−fraction cleaved)^(1/2))

where the fraction cleaved is the sum of the cleavage product peaksdivided by the sum of the cleavage product and parent peaks.⁵

VIII. Sanger Sequencing to Detect Genome Editing.

Purified PCR amplicons of the T7EI assay together with their forwardprimers were sequenced by The McDermott Center Sequencing core facilityin UT Southwestern Medical Center. The sequencing data were finallyanalyzed using online analysis software, ICE Analysis, which is a webtool provided by Synthego.

IX. In Vitro Gene Editing in Hela-GFP Cells.

HeLa-GFP reporter cells were cultured in DMEM containing 10% FBS and 1%penicillin/streptomycin at 37° C./5% CO₂. For the experiments, HeLa-GFPcells were seeded into 12-well plates at a cell density of 1.5×10⁵ cellsper well and incubated overnight. Then, the medium was replaced with 0.5mL of fresh complete DMEM and 100 uL of nanoparticle dispersion wereadded (the final concentration of sgRNA was fixed at 24 nM). Three daysafter treatment, the cells were analyzed using a fluorescence microscope(Keyence). For the flow cytometry analysis, the cells were collected,washed with PBS, re-suspended in PBS, and analyzed using a BD AnalyzersLSRFortessa SORP (BD Biosciences).

X. Stability of 5A2-DOT-10 Cas9/sgGFP.

To measure stability, 5A2-DOT-10 LNPs encapsulating Cas9/sgGFP RNPcomplexes were prepared and stored them at 4° C. for 2 months. The sizeand PDI of these nanoparticles were tested after storing for differenttimes and their gene editing efficiency were also evaluated in HeLa-GFPcells by adding nanoparticles (24 nM sgRNA dose) and quantifying geneediting after 3 days. For each time point, an aliquot of stored5A2-DOT-10 LNPs encapsulating Cas9/sgGFP RNPs was taken and analyzed(size, PDI, efficacy).

XI. Animal Experiments.

All animal experiments were approved by the Institution Animal Care andUse Committees of The University of Texas Southwestern Medical Centerand were consistent with local, state and federal regulations asapplicable. C57BL/6 mice were obtained from the UTSW Mouse Breeding CoreFacility. B6.Cg-Gt(ROSA)26Sor^(tm9(CAG-tdTomato)Hze)/J mice (also knownas Ai9 or Ai9(RCL-tdT) mice) were obtained from The Jackson Laboratory(007909) and bred to maintain homozygous expression of the Cre reporterallele that has a loxP-flanked STOP cassette preventing transcription ofa CAG promoter-driven red fluorescent tdTomato protein. FollowingCas9/sgRNA RNPs mediated gene editing, Ai9 mice will express tdTomatofluorescence. Ai9 mice are congenic on the C57BL/6J genetic background.

XII. In Vivo Gene Editing.

For gene editing in muscles, Td-Tomato mice were injected with5A2-DOT-10 LNPs encapsulating Cas9/sgTOM RNP complexes at dose of 1mg/kg of sgTOM in the left leg by intra-muscle injection. RNAiMAXencapsulating Cas9/sgTOM RNP complexes was used as positive control.After treatment for 7 days, the muscle tissues of all treatment groupswere collected and imaged using an IVIS Lumina system (Perkin Elmer).Afterwards, the muscle tissues were embedded in optimal cuttingtemperature (OCT) compound and cut into 10 μm slices. The sections werefixed with 4% Paraformaldehyde (Thermo Fisher Scientific) for 20 min,washed three times using PBS buffer. Afterwards, one drop of ProLongGold Mountant with DAPI (Thermo Fisher Scientific) was applied onto eachslide. A coverslip was placed, and the slides were imaged by confocalmicroscopy (Zeiss LSM 700). For gene editing in brain, Td-Tomato micewere injected with 5A2-DOT-10 LNPs encapsulating Cas9/sgTOM RNPcomplexes at dose of 0.15 mg/kg of sgTOM by intra-brain injection. Aftertreatment for 6 days, the brains were excised and imaged using IVISLumina system. Frozen sections of brains were prepared as the protocolmentioned above and imaged by confocal microscopy.

For gene editing by i.v. injection, Td-Tomato mice were treated with5A2-DOT-X LNPs containing different percentage of DOTAP at dose of 1.5mg/kg of sgTOM by tail vein injection. After treatment for 7 days, allorgans were collected and imaged using IVIS Lumina system. The frozensections of these tissues were prepared as the protocol mentioned aboveand imaged by confocal microscopy.

XIII. PCR for Eml4-Alk Rearrangements.

The in vivo Eml4-Alk rearrangements were tested by nested PCR (Blasco etal., 2014). For the first round PCR, 40 ng of genomic DNA was used astemplate with PCR program of 95° C. for 5 min; (95° C. for 30 sec; 64°C. for 30 sec; 72° C. for 30 sec) for 18 cycles; 72° C. for 7 min andthen keep at 4° C. For the second round PCR, 1 μl of the 1^(st) roundPCR product (100 dilutions) was used for PCR reactions (95° C. for 5min; (95° C. for 30 sec; 68° C. for 30 sec; 72° C. for 30 sec) for 30cycles; 72° C. for 7 min and then keep at 4° C. Primers used in the PCRreactions are listed in Table 6.

TABLE 6  Listing of Primers Forward Primers  Reverse Primers Name(5′ to 3′) (5′ to 3′) LUC ATGGAAGACGCCAAAAACATA AACACTTAAAATCGCAAGAAAGGCCCGGCGCCATTC AGTATCCGGAATG (SEQ ID NO: 26) (SEQ ID NO: 27) GFPGTGGTGCCCATCCTGGTCGAG CGCTTCTCGTTGGGG (SEQ ID NO: 28) TCTTTGC(SEQ ID NO: 29) PTEN ATCCGTCTTCTCCCCATTCCG GACGAGCTCGCTAAT(SEQ ID NO: 30) CCAGTG (SEQ ID NO: 31) P53 ATAGAGACGCTGAGTCCGGTTCCTAAGCCCAAGAGG C (SEQ ID NO: 32) AAACAGA (SEQ ID NO: 33) RB1CTGTGCTGGTGTGTGCAAACT CTGTCACAGTGAAACT ATA (SEQ ID NO: 34)CGTTACTTTGTATATC (SEQ ID NO: 35) Eml4 ACAAGGCTCTGGCTTCCATTGGATCAAAGCAAGGCC (SEQ ID NO: 36) TTGTGCAT (SEQ ID NO: 37) AlkTCTGAGCCCCTTCCATCTGAC AGCTCAGCAGAAGCT C (SEQ ID NO: 38) CAGCAG(SEQ ID NO: 39) Eml4-Alk CCCAGTCATCAGTTGCTATGC GGGTTTCCTTTGGTT inversionAATT (SEQ ID NO: 40) CACAGATCCA (1^(st) round) (SEQ ID NO: 41) Eml4-AlkCGTTTTTCCACAAGAGCTAAG GTGGTTTGGTCACAT inversion GCT (SEQ ID NO: 42)CTCAGGTG (2^(nd) round) (SEQ ID NO: 43)

XIV. H&E Staining and Immunohistochemistry (IHC).

Briefly, 10% Formalin solution fixed tissues were embedded in paraffin,sectioned and H&E stained by the Molecular Pathology Core at UTSW. The 4μm sections were performed in the standard fashion and detected withElite ABC Kit and DAB Substrate (Vector Laboratories) for IHC.

XV. Statistical Analyses.

Statistical analyses were conducted using two-sided Student's t-test byGraphPad Prism software, version 7.04 (GraphPad Software, USA). Noadjustments were made in any statistical test. A P value <0.05 wasconsidered statistically significant.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this disclosure have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the disclosure. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of thedisclosure as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A method for non-liver targeted delivery of atherapeutic agent, the method comprising: administering to a subject inneed thereof said therapeutic agent assembled with a selective organtargeting (SORT) lipid composition, which SORT lipid compositioncomprises: (i) an ionizable cationic lipid, and (ii) a SORT lipidseparate from said ionizable cationic lipid, which SORT lipid isselected from the group consisting of a cationic SORT lipid, azwitterionic SORT lipid, and an anionic SORT lipid; wherein, upon saidadministering, a surface of said SORT lipid composition interacts withapolipoprotein E (Apo E) to a lesser degree than with an endogenousprotein that is not Apo E in said subject, thereby providing a lesseramount or activity of said therapeutic agent in liver or a cell thereinin said subject as compared to that achieved absent said SORT lipid. 2.The method of claim 1, wherein, upon said administering, said endogenousprotein binds to said surface of said SORT lipid composition in saidsubject at a greater amount than Apo E.
 3. The method of claim 1,wherein said endogenous protein is vitronectin, beta-2 glycoprotein 1,or apolipoprotein H (Apo H).
 4. The method of claim 1, wherein saidendogenous protein is beta-2 glycoprotein 1 or apolipoprotein H (Apo H).5. The method of claim 1, wherein said endogenous protein is not beta-2glycoprotein 1 or apolipoprotein H (Apo H); and wherein said surface ofsaid SORT lipid composition interacts with Apo H to a lesser degree thanwith said endogenous protein in said subject.
 6. The method of claim 5,wherein said endogenous protein is vitronectin.
 7. The method of claim1, wherein said administering comprises systemic administration.
 8. Themethod of claim 1, wherein said administering comprises intravenousadministration.
 9. The method of claim 1, wherein the method provides agreater amount or activity of said therapeutic agent in a non-liverorgan or a cell therein in said subject as compared to that achievedabsent said SORT lipid.
 10. The method of claim 9, wherein saidnon-liver organ or said cell therein comprises a lung, spleen, bonemarrow, a lymph node, or a cell in any of the aforementioned organs. 11.The method of claim 10, wherein said non-liver organ or said celltherein comprises a lung, or a cell therein.
 12. The method of claim 10,wherein said non-liver organ or said cell therein comprises spleen, bonemarrow, a lymph node, or a cell in any of the aforementioned organs. 13.The method of claim 1, wherein said SORT lipid is present in said SORTlipid composition at a molar percentage from about 5% to about 65%. 14.The method of claim 1, wherein said cationic SORT lipid is a permanentlycationic SORT lipid or an ionizable cationic SORT lipid.
 15. The methodof claim 1, wherein said cationic SORT lipid is a permanently cationicSORT lipid comprising a quaternary ammonium ion.
 16. The method of claim1, wherein said cationic SORT lipid is a permanently cationic SORT lipidhaving a structure of Formula (I), Formula (II), or Formula (III), or apharmaceutically acceptable salt, stereoisomer, tautomer thereof:

wherein, in Formula (I): R₁ and R₂ are each independentlyalkyl_((C8-C24)), alkenyl_((C8-C24)), or a substituted version of eithergroup; R₃, R₃′, and R₃″ are each independently alkyl_((C≤6)); orsubstituted alkyl_((C≤6)); and X⁻ is a monovalent anion; or

wherein, in Formula (II): R₄ and R₄′ are each independentlyalkyl_((C6-C24)), alkenyl_((C6-C24)), or a substituted version of eithergroup; R₄″ is alkyl_((C≤24)), alkenyl_((C≤24)), or a substituted versionof either group; R₄′″ is alkyl_((C1-C8)), alkenyl_((C2-C8)), or asubstituted version of either group; and X₂ ⁻ is a monovalent anion; or

wherein, in Formula (III): R₁ and R₂ are each independentlyalkyl_((C8-C24)), alkenyl_((C8-C24)), or a substituted version of eithergroup; R₃, R₃′, and R₃″ are each independently alkyl_((C≤6)) orsubstituted alkyl_((C≤6)); R₄ is alkyl_((C≤6)) or substitutedalkyl_((C≤6)); and X⁻ is a monovalent anion.
 17. The method of claim 1,wherein said zwitterionic SORT lipid is a zwitterionic phospholipid. 18.The method of claim 1, wherein said anionic SORT lipid comprises aphosphate group.
 19. The method of claim 1, wherein said anionic SORTlipid has a structure of Formula (IV):

wherein, in Formula (IV): R₁ and R₂ are each independentlyalkyl_((C8-C24)), alkenyl_((C8-C24)), or a substituted version of eithergroup; R₃ is hydrogen, alkyl_((C≤6)), or substituted alkyl_((C≤6)), or—Y₁—R₄, wherein: Y₁ is alkanediyl_((C≤6)) or substitutedalkanediyl_((C≤6)); and R₄ is acyloxy_((C≤8-24)) or substitutedacyloxy_((C≤8-24)).
 20. The method of claim 1, wherein said cationicSORT lipid is 1,2-dioleoyl-3-dimethylammonium-propane (DODAP).
 21. Themethod of claim 1, wherein said ionizable cationic lipid is present insaid SORT lipid composition at a molar percentage from about 5% to about30%.
 22. The method of claim 1, wherein said ionizable cationic lipid isa dendrimer or dendron, or a pharmaceutically acceptable salt thereof.23. The method of claim 1, wherein said SORT lipid composition furthercomprises a phospholipid, a polymer-conjugated lipid, a steroid orsteroid derivative, or a combination thereof separate from said SORTlipid.
 24. The method of claim 1, wherein said SORT lipid compositionfurther comprises a polymer-conjugated lipid separate from said SORTlipid.
 25. The method of claim 24, wherein said polymer-conjugated lipidcomprises one or more hydrophobic moieties each comprising at least 6carbon atoms.
 26. The method of claim 24, wherein saidpolymer-conjugated lipid comprises one or more hydrophobic moieties eachcomprising 6 to 24 carbon atoms.
 27. The method of claim 24, whereinsaid polymer-conjugated lipid comprises a polymer component having anaverage molecular weight from about 100 Daltons (Da) to about 15,000Daltons.
 28. The method of claim 24, wherein said polymer-conjugatedlipid is a polyethylene glycol (PEG) conjugated lipid.
 29. The method ofclaim 1, wherein said therapeutic agent comprises a small interferingribonucleic acid (siRNA), a micro-ribonucleic acid (miRNA), a primarymicro-ribonucleic acid (pri-miRNA), a messenger ribonucleic acid (mRNA),a clustered regularly interspaced short palindromic repeats (CRISPR)related nucleic acid, a CRISPR-RNA (crRNA), a single guide ribonucleicacid (sgRNA), a trans-activating CRISPR ribonucleic acid (tracrRNA), aplasmid deoxyribonucleic acid (pDNA), a transfer ribonucleic acid(tRNA), an antisense oligonucleotide (ASO), a guide ribonucleic acid, adouble stranded deoxyribonucleic acid (dsDNA), a single strandeddeoxyribonucleic acid (ssDNA), a single stranded ribonucleic acid(ssRNA), a double stranded ribonucleic acid (dsRNA), a protein, aCRISPR-associated (Cas) protein, or a combination thereof.
 30. Themethod of claim 1, wherein said therapeutic agent is present at a ratioto said SORT lipid composition of about 1:1 to about 1:100.